AU2019220495B2 - Methods for treating cancer with anti-PD-1 antibodies - Google Patents

Abstract

The present invention relates to methods for treating cancer in a patient comprising administering a PD-1 antagonist, e.g., an anti-PD-1 antibody or antigen binding fragment thereof (e.g. pembrolizumab), in specific amounts to the patient about every six weeks. In some embodiments, the amount of anti-PD-1 antibody or antigen binding fragment thereof is about 400 mg. In certain embodiments, the PD-1 antagonist is pembrolizumab, or an antigen binding fragment thereof. Also provided are compositions and kits comprising a dosage of an anti-PD-1 antibody, or antigen-binding fragment thereof, and uses thereof for treating cancer.

Description

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number

(43) International Publication Date WO 2019/160751 A2 22 August 2019 (22.08.2019) WIPOIPCT WIPOIPCT (51) International Patent Classification: (US). JAIN, Lokesh [IN/US]; 126 East Lincoln Avenue, A61K 39/395 (2006.01) Rahway, New Jersey 07065-0907 (US). LI, Mengyao

[CN/US]; 42 Forest Drive, Apt. B, Springfield, New Jersey (21) International Application Number: 07084 (US). PCT/US2019/017177

(22) International Filing Date: (74) Common Representative: MERCK SHARP & DOHME CORP.; 126 East Lincoln Avenue, Rahway, New Jersey 08 February 2019 (08.02.2019) 07065-0907 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/630,038 13 February 2018 (13.02.2018) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 62/732,828 18 September 2018 (18.09.2018) US HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (71) Applicant: MERCK SHARP & DOHME CORP.

[US/US]; 126 East Lincoln Avenue, Rahway, New Jersey MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 07065-0907 (US). OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (72) Inventors; and TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants (for US only): LALA, Mallika [IN/US]; 126 East Lincoln Avenue, Rahway, New Jersey 07065-0907

(54) Title: METHODS FOR TREATING CANCER WITH ANTI-PD-1 ANTIBODIES

10,000

1,000 Cmax,ss (µg/mL)

100

10 10 WO 2019/160751 A2

2 mpk Q3W 200 mg Q3W 400 mg Q6W 10 mpk Q2W

FIG.2

(57) Abstract: The present invention relates to methods for treating cancer in a patient comprising administering a PD-1 antagonist, e.g., an anti-PD-1 antibody or antigen binding fragment thereof (e.g. pembrolizumab), in specific amounts to the patient about every six weeks. In some embodiments, the amount of anti-PD-1 antibody or antigen binding fragment thereof is about 400 mg. In certain embodiments, the PD-1 antagonist is pembrolizumab, or an antigen binding fragment thereof. Also provided are compositions and kits comprising a dosage of an anti-PD-1 antibody, or antigen-binding fragment thereof, and uses thereof for treating cancer.

[Continued on next page]

WO 2019/160751 A2 (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).

Declarations under Rule 4.17: as to applicant's entitlement to apply for and be granted a

- patent (Rule 4.17(ii))

as to the applicant's entitlement to claim the priority of the

- earlier application (Rule 4.17(iii))

Published: without international search report and to be republished

- upon receipt of that report (Rule 48.2(g))

with sequence listing part of description (Rule 5.2(a))

-

TITLE OF THE INVENTION METHODS FOR TREATING CANCER WITH ANTI-PD-1 ANTIBODIES

FIELD OF THE INVENTION The present invention relates to therapies useful for the treatment of cancer. In

particular, the invention relates to a method for treating cancer which comprises administering to

a patient in need thereof an anti-PD-1 antibody, or antigen binding fragment thereof, using the

dosage regimens specified herein.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application number

62/630,038, filed February 13, 2018, and U.S. provisional application number 62/732,828, filed

September 18, 2018, the contents of which are hereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The sequence listing of the present application is submitted electronically via

EFS-Web as an ASCII formatted sequence listing with a file name "24567WOPCT-SEQLIST- 25JAN2019.TXT" creation date of January 25, 2019, and a size of 23.7 kb. This sequence

listing submitted via EFS-Web is part of the specification and is herein incorporated by reference

in its entirety.

BACKGROUND OF THE INVENTION PD-1 is recognized as an important player in immune regulation and the

maintenance of peripheral tolerance. PD-1 is moderately expressed on naive T, B and NKT cells

and up-regulated by T/B cell receptor signaling on lymphocytes, monocytes and myeloid cells

(Sharpe et al., The function of programmed cell death 1 and its ligands in regulating

autoimmunity and infection. Nature Immunology (2007); 8:239-245).

Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed

in human cancers arising in various tissues. In large sample sets of e.g. ovarian, renal, colorectal,

pancreatic, liver cancers and melanoma, it was shown that PD-L1 expression correlated with

poor prognosis and reduced overall survival irrespective of subsequent treatment (Dong et al.,

Nat Med. 8(8):793-800 (2002); Yang et al. Invest Ophthalmol Vis Sci. 49: 2518-2525 (2008);

Ghebeh et al. Neoplasia 8:190-198 (2006); Hamanishi et al., Proc. Natl. Acad. Sci. USA 104:

3360-3365 (2007); Thompson et al., Cancer 5: 206-211 (2006) ; Nomi et al., Clin. Cancer

Research 13:2151-2157 (2007); Ohigashi et al., Clin. Cancer Research 11: 2947-2953 (2005);

Inman et al., Cancer 109: 1499-1505 (2007); Shimauchi et al. Int. J. Cancer 121:2585-2590

(2007); Gao et al. Clin. Cancer Research 15: 971-979 (2009); Nakanishi J. Cancer Immunol

Immunother. 56: 1173- 1182 (2007); and Hino et al., Cancer 00: 1-9 (2010)).

2 16 Jan 2025 2019220495 16 Jan 2025

Similarly, PD-1 expression on tumor infiltrating lymphocytes was found to mark dysfunctional T cells in breast cancer and melanoma (Ghebeh et al, BMC Cancer. 2008 8:5714- 15 (2008); Ahmadzadeh et al., Blood 114: 1537-1544 (2009)) and to correlate with poor prognosis in renal cancer (Thompson et al., Clinical Cancer Research 15: 1757-1761(2007)). Thus, it has been proposed that PD-L1 expressing tumor cells interact with PD-1 expressing T cells to attenuate T cell activation and evasion of immune surveillance, thereby contributing to 2019220495

an impaired immune response against the tumor. Immune checkpoint therapies targeting the PD-1 axis have resulted in groundbreaking improvements in clinical response in multiple human cancers (Brahmer et al., N Engl J Med 2012, 366: 2455-65; Garon et al. N Engl J Med 2015, 372: 2018-28; Hamid et al., N Engl J Med 2013, 369: 134-44; Robert et al., Lancet 2014, 384: 1109-17; Robert et al., N Engl J Med 2015, 372: 2521-32; Robert et al., N Engl J Med 2015, 372: 320-30; Topalian et al., N Engl J Med 2012, 366: 2443-54; Topalian et al., J Clin Oncol 2014, 32: 1020-30; Wolchok et al., N Engl J Med 2013, 369: 122-33). Immune therapies targeting the PD-1 axis include monoclonal antibodies directed to the PD-1 receptor (KEYTRUDA™ (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ, USA and OPDIVO™ (nivolumab), Bristol-Myers Squibb Company, Princeton, NJ, USA) and also those that bind to the PD-L1 ligand (MPDL3280A; TECENTRIQ™ (atezolizumab), Genentech, San Francisco, CA, USA; IMFINZI™ (durvalumab), AstraZeneca Pharmaceuticals LP, Wilmington, DE; BAVENCIO™ (avelumab), Merck KGaA, Darmstadt, Germany). Both therapeutic approaches have demonstrated anti-tumor effects in numerous cancer types. It would be beneficial to develop additional dosing schedules that allow for the administration of a safe and effective dose of an anti-PD-1 antibody that is more convenient for patients.

SUMMARYOF SUMMARY OFTHE THE INVENTION INVENTION In a first aspect, the present invention provides a method of treating cancer in a human patient comprising administering about 400 mg of pembrolizumab to the patient every approximately six weeks. In a second aspect, the present invention provides a use of about 400 mg of pembrolizumab in the manufacture of a medicament for treating cancer in a human patient, wherein the medicament is administered to the patient every approximately six weeks.

2a 20 Oct 2025

In a third aspect, the present invention provides a composition comprising about 400 mg of pembrolizumab and a pharmaceutically acceptable carrier when used approximately every six weeks for the treatment of cancer.

In a fourth aspect, the present invention provides the use of the composition of the third 2019220495

aspect for treating an individual suffering from cancer.

The present invention also provides alternative, less frequent, dosing regimens for treating a cancer patient with an anti-PD-1 antibody, or antigen-binding fragment thereof, wherein the dosing schedule is expected to provide a safe and effective dose of the anti-PD-1 antibody, or antigen-binding fragment thereof. specifically, the invention provides a method of treating cancer in a human patient comprising administering about 400 mg of an anti-PD-1 antibody or antigen binding fragment thereof to the patient every six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) light chain complementarity determining regions (CDRS) comprising a sequence of amino acids as set forth in SEQ ID NOs: 1, 2 and 3 and heavy chain CDRS comprising a sequence of amino acids as set forth in SEQ ID NOs: 6, 7 and 8; or (b) light chain CDRS comprising a sequence of amino acids as set forth in SEQ ID NOs: 11, 12 and 13 and heavy chain CDRS comprising a sequence of amino acids as set forth in SEQ ID NOs: 14, 15 and 16. In preferred embodiments of the invention, the antibody or antigen-binding fragment is pembrolizumab.

WO wo 2019/160751 PCT/US2019/017177

In embodiments of the invention, the amount of anti-PD-1 antibody or antigen-

binding fragment thereof administered to the patient is from about 350 mg to about 450 mg. In

further embodiments, the amount of antibody or antigen-binding fragment is about 400 mg. In

further embodiments, the amount of antibody or antigen-binding fragment is 400 mg.

In all of the above treatment methods, compositions and uses herein, the PD-1

antibody or antigen-binding fragment inhibits the binding of PD-L1 to PD-1, and preferably also

inhibits the binding of PD-L2 to PD-1. In some preferred embodiments of the treatment

methods, compositions and uses of the invention, the PD-1 antibody or antigen-binding fragment

is a monoclonal antibody, which specifically binds to PD-1 and blocks the binding of PD-L1 to

PD-1. In one particular embodiment, the anti-PD-1 antibody comprises a heavy chain and a light

chain, and wherein the heavy and light chains comprise the amino acid sequences shown in

Figure 1 (SEQ ID NO:5 and SEQ ID NO: In some embodiments of any of the above treatment methods, compositions and

uses, the cancer expresses one or both of PD-L1 and PD-L2. In some embodiments, PD-L1

expression is elevated in the cancer.

In certain embodiments of any of the methods described herein, the anti-PD-1

antibody or antigen binding fragment is administered to a patient subcutaneously.

In alternative embodiments of any of the methods described herein, the anti-PD-1

antibody or antigen binding fragment is administered to a patient intravenously.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows amino acid sequences of the light chain and heavy chain for an

exemplary anti-PD-1 monoclonal antibody useful in the present invention (SEQ ID NOs:5 and

10, respectively). Light chain and heavy chain variable regions are underlined (SEQ ID NO's 4

and 9) and CDRs are bold and boxed.

FIGURE 2 shows that pembrolizumab Cmax at steady state for 400 mg Q6W lies

within the range from 2 mg/kg Q3W and 200 mg Q3W to 10 mg/kg Q2W. FIGURE 3 shows that pembrolizumab exposures (Cavg and Cmin) at steady state

are similar for 400 mg Q6W relative to 2 mg/kg Q3W and 200 mg Q3W.

FIGURE 4A and 4B show the pembrolizumab pharmacokinetic profiles at steady

state for the 400 mg Q6W dosing regimen compared to the 200 mg, Q3W, flat dosing regimen

(top), the Q3W, 2 mg/kg weight-based dosing regimen (middle), and the Q2W, 10 mg/kg weight-

based dosing regimen (bottom). Results are provided for log scale concentrations (FIG. 4A) and

linear scale concentrations (FIG. 4B).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Abbreviations

As used throughout the specification and appended claims, the following

abbreviations apply:

WO wo 2019/160751 PCT/US2019/017177

adverse event AE area under the concentration-time curve at steady state AUCss BICR blinded independent central review

Cavg,ss time averaged concentration at steady state

complementarity determining region CDR CI confidence interval

Cmax,ss peak concentrations at steady state

Cmin,ss trough concentrations at steady state

CPS combined positive score

duration of response DOR electrocardiogram ECG Eastern Cooperative Oncology Group ECOG E-R exposure (concentration)-response

FFPE formalin-fixed paraffin-embedded

framework region FR geometric mean GM hepatocellular carcinoma HCC head and neck squamous cell cancer HNSCC Hodgkin lymphoma HL IgG immunoglobulin G

IHC immunohistochemistry or immunohistochemical

IV intravenous

LPS lymphoma proportion score

monoclonal antibody mAb Merkel cell carcinoma MCC MEL melanoma mismatch repair MMR modified proportion score MPS magnetic resonance imaging MRI microsatellite instability-high MSI-H NCICTCAE National Cancer Institute - Common Terminology Criteria for Adverse Events

non-small cell lung cancer NSCLC objective response rate ORR overall survival os progressive disease PD PD-1 programmed death 1 (a.k.a. programmed cell death-1 and

programmed death receptor 1)

PD-L1 programmed cell death 1 ligand 1

WO wo 2019/160751 PCT/US2019/017177

PD-L2 programmed cell death 1 ligand 2

progression free survival PFS pharmacokinetic PK one dose every two weeks Q2W one dose every three weeks Q3W one dose every six weeks Q6W renal cell carcinoma RCC serious adverse event SAE SC subcutaneous

TPS tumor proportion score

VH immunoglobulin heavy chain variable region

VL immunoglobulin light chain variable region

So that the invention may be more readily understood, certain technical and

scientific terms are specifically defined below. Unless specifically defined elsewhere in this

document, all other technical and scientific terms used herein have the meaning commonly

understood by one of ordinary skill in the art to which this invention belongs.

Reference to "or" indicates either or both possibilities unless the context clearly

dictates one of the indicated possibilities. In some cases, "and/or" was employed to highlight

either or both possibilities.

As used herein, including the appended claims, the singular forms of words such

as "a," "an," and "the," include their corresponding plural references unless the context clearly

dictates otherwise.

The term "about", when modifying the quantity (e.g., mg) of a substance or

composition, or the value of a parameter characterizing a step in a method, or the like, refers to

variation in the numerical quantity that can occur, for example, through typical measuring,

handling and sampling procedures involved in the preparation, characterization and/or use of the

substance or composition; through inadvertent error in these procedures; through differences in

the manufacture, source, or purity of the ingredients employed to make or use the compositions

or carry out the procedures; and the like. In certain embodiments, "about" can mean a variation

of 0.1%, 0.5%, 1%, 2%, 3%, 4%, 1 5%, 6%, 7%, 8%, 9% or 10%. When referring to the dosage of "about 400 mg," the dosage can be from 360 mg to 440 mg, from 370

mg to 430 mg, from 380 mg to 420 mg, from 390 mg to 410 mg, from 395 mg to 405 mg, from

400 mg to 440 mg, or from 390 mg to 440 mg. It alternative embodiments, the dosage can be

360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg,

415 mg, 420 mg, 425 mg, 430 mg, 435 mg, or 440 mg. When referring to the amount of time

between administrations in a therapeutic treatment regimen (i.e., amount of time between

administrations of the anti-PD-1 antibody or antigen binding fragment thereof, e.g. "about 6

weeks," which is used interchangeably herein with "approximately every six weeks"), "about"

5

WO wo 2019/160751 PCT/US2019/017177

refers to the stated time a variation that can occur due to patient/clinician scheduling and

availability around the 6-week target date. For example, "about 6 weeks" can refer to 6 weeks +5

days, 6 weeks +4 days, 6 weeks +3 days, 6 weeks +2 days or 6 weeks +1 day, or may refer to 5

weeks, 2 days through 6 weeks, 5 days.

Pharmacokinetic "steady state" is a period of time during which any accumulation

of drug concentrations owing to multiple doses has been maximized and systemic drug exposure

is considered uniform after each subsequent dose administered; in the specific case of

pembrolizumab, steady state is achieved at and after ~16 weeks of administration.

AUCss, Cavg,ss and Cmin,ss are pharmacokinetic measures of the systemic

exposure to the drug (e.g. pembrolizumab) in humans after its administration, and are typically

considered drivers of drug efficacy. AUCss and Cavg,ss represent the average exposure over a

dosing interval, but differ in terms of units. "Cmin,ss" represents the minimum or lowest

(trough) drug concentration observed at the end of a dosing interval, just before the next dose is

administered.

"Cmax,ss" is the maximum or highest (peak) drug concentration observed soon

after its administration. In the specific case of pembrolizumab, which is administered as

intravenous infusion, the peak concentration occurs immediately after end of infusion. Cmax,ss

is a metric that is typically considered a driver of driver safety.

"Administration" and "treatment," as it applies to an animal, human, experimental

subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical,

therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or

biological fluid. "Treat" or "treating" a cancer, as used herein, means to administer an anti-PD-1

antibody, or antigen-binding fragment, to a subject having a cancer, or diagnosed with a cancer,

to achieve at least one positive therapeutic effect, such as for example, reduced number of cancer

cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or

reduced rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the

following: inducing/increasing an antitumor immune response, decreasing the number of one or

more tumor markers, halting or delaying the growth of a tumor or blood cancer or progression of

disease associated with PD-1 binding to its ligands PD-L1 and/or PD-L2 ("PD-1-related

disease") such as cancer, stabilization of PD-1-related disease, inhibiting the growth or survival

of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors,

decreasing the level of one or more tumor markers, ameliorating or abrogating the clinical

manifestations of PD-1-related disease, reducing the severity or duration of the clinical

symptoms of PD-1-related disease such as cancer, prolonging the survival of a patient relative to

the expected survival in a similar untreated patient, and inducing complete or partial remission of

a cancerous condition or other PD-1 related disease.

Positive therapeutic effects in cancer can be measured in a number of ways (See,

W. A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumor growth

inhibition, according to NCI standards, a T/C < 42% is the minimum level of anti-tumor activity.

WO wo 2019/160751 PCT/US2019/017177

A T/C < 10% is considered a high anti-tumor activity level, with T/C (%) : Median tumor

volume of the treated/Median tumor volume of the control X 100. In some embodiments, the

treatment achieved by a therapeutically effective amount is any of progression free survival

(PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as "Time to

Tumor Progression" indicates the length of time during and after treatment that the cancer does

not grow, and includes the amount of time patients have experienced a complete response or a

partial response, as well as the amount of time patients have experienced stable disease. DFS

refers to the length of time during and after treatment that the patient remains free of disease. os

refers to a prolongation in life expectancy as compared to naive or untreated individuals or

patients. While an embodiment of the treatment methods, compositions and uses of the present

invention may not be effective in achieving a positive therapeutic effect in every patient, it

should do SO in a statistically significant number of subjects as determined by any statistical test

known in the art such as the Student's t-test, the chi -test, the U-test according to Mann and

Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.

The term "patient" (alternatively referred to as "subject" or "individual" herein)

refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) capable of being treated with the methods

and compositions of the invention, most preferably a human. In some embodiments, the patient

is an adult patient. In other embodiments, the patient is a pediatric patient.

The term "antibody" refers to any form of antibody that exhibits the desired

biological or binding activity. Thus, it is used in the broadest sense and specifically covers, but

is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal

antibodies, humanized, fully human antibodies, and chimeric antibodies. "Parental antibodies"

are antibodies obtained by exposure of an immune system to an antigen prior to modification of

the antibodies for an intended use, such as humanization of an antibody for use as a human

therapeutic.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer

includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa)

and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a

variable region of about 100 to 110 or more amino acids primarily responsible for antigen

recognition. The carboxy-terminal portion of the heavy chain may define a constant region

primarily responsible for effector function. Typically, human light chains are classified as kappa

and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta,

gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,

respectively. Within light and heavy chains, the variable and constant regions are joined by a "J"

region of about 12 or more amino acids, with the heavy chain also including a "D" region of

about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd

ed. Raven Press, N.Y. (1989).

WO wo 2019/160751 PCT/US2019/017177

The variable regions of each light/heavy chain pair form the antibody binding site.

Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific

antibodies, the two binding sites are, in general, the same.

Typically, the variable domains of both the heavy and light chains comprise three

hypervariable regions, also called complementarity determining regions (CDRs), which are

located within relatively conserved framework regions (FR). The CDRs are usually aligned by

the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-

terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3,

CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with

the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National

Institutes of Health, Bethesda, Md. ; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv.

Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987)

JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

The term "hypervariable region" refers to the amino acid residues of an antibody

that are responsible for antigen-binding. The hypervariable region comprises amino acid

residues from a "complementarity determining region" or "CDR" (i.e. CDRL1, CDRL2 and

CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain

variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th

Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR

regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-

917 (defining the CDR regions of an antibody by structure). The term "framework" or "FR"

residues refers to those variable domain residues other than the hypervariable region residues

defined herein as CDR residues.

Unless otherwise indicated, an "antibody fragment" or "antigen binding fragment"

refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to

specifically bind to the antigen bound by the full-length antibody, e.g. fragments that retain one

or more CDR regions. Examples of antibody binding fragments include, but are not limited to,

Fab, Fab', F(ab')2, and Fv fragments.

An antibody that "specifically binds to" a specified target protein is an antibody

that exhibits preferential binding to that target as compared to other proteins, but this specificity

does not require absolute binding specificity. An antibody is considered "specific" for its

intended target if its binding is determinative of the presence of the target protein in a sample,

e.g. without producing undesired results such as false positives. Antibodies, or binding fragments

thereof, useful in the present invention will bind to the target protein with an affinity that is at

least two fold greater, preferably at least ten times greater, more preferably at least 20-times

greater, and most preferably at least 100-times greater than the affinity with non-target proteins.

As used herein, an antibody is said to bind specifically to a polypeptide comprising a given

amino acid sequence, e.g. the amino acid sequence of a mature human PD-1 or human PD-L1

-

WO wo 2019/160751 PCT/US2019/017177

molecule, if it binds to polypeptides comprising that sequence but does not bind to proteins

lacking that sequence.

"Chimeric antibody" refers to an antibody in which a portion of the heavy and/or

light chain is identical with or homologous to corresponding sequences in an antibody derived

from a particular species (e.g., human) or belonging to a particular antibody class or subclass,

while the remainder of the chain(s) is identical with or homologous to corresponding sequences

in an antibody derived from another species (e.g., mouse) or belonging to another antibody class

or subclass, as well as fragments of such antibodies, SO long as they exhibit the desired biological

activity.

"Human antibody" refers to an antibody that comprises human immunoglobulin

protein sequences only. A human antibody may contain murine carbohydrate chains if produced

in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "mouse

antibody" or "rat antibody" refer to an antibody that comprises only mouse or rat

immunoglobulin sequences, respectively.

"Humanized antibody" refers to forms of antibodies that contain sequences from

non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain

minimal sequence derived from non-human immunoglobulin. In general, the humanized

antibody will comprise substantially all of at least one, and typically two, variable domains, in

which all or substantially all of the hypervariable loops correspond to those of a non-human

immunoglobulin and all or substantially all of the FR regions are those of a human

immunoglobulin sequence. The humanized antibody optionally also will comprise at least a

portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

The prefix "hum", "hu" or "h" is added to antibody clone designations when necessary to

distinguish humanized antibodies from parental rodent antibodies. The humanized forms of

rodent antibodies will generally comprise the same CDR sequences of the parental rodent

antibodies, although certain amino acid substitutions may be included to increase affinity,

increase stability of the humanized antibody, or for other reasons.

The terms "cancer", "cancerous", or "malignant" refer to or describe the

physiological condition in mammals that is typically characterized by unregulated cell growth.

Examples of cancer include but are not limited to, carcinoma, lymphoma, leukemia, blastoma,

and sarcoma. More particular examples of such cancers include, but are not limited to, squamous

cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin

lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma,

gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia,

lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer,

thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma

multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast

cancer, colon carcinoma, and head and neck cancer. Additional cancers that may be treated in

WO wo 2019/160751 PCT/US2019/017177

accordance with the present invention include those characterized by elevated expression of one

or both of PD-L1 and PD-L2 in tested tissue samples.

"Biotherapeutic agent" means a biological molecule, such as an antibody or

fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports

tumor maintenance and/or growth or suppresses the anti-tumor immune response.

"CDR" or "CDRs" means complementarity determining region(s) in an

immunoglobulin variable region, generally defined using the Kabat numbering system.

"Platinum-containing chemotherapy" (also known as platins) refers to the use of

chemotherapeutic agent(s) used to treat cancer that are coordination complexes of platinum.

Platinum-containing chemotherapeutic agents are alkylating agents that crosslink DNA, resulting

in ineffective DNA mismatch repair and generally leading to apoptosis. Examples of platins

include cisplatin, carboplatin, and oxaliplatin.

"Chemotherapeutic agent" is a chemical compound useful in the treatment of

cancer. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents,

antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics,

topisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor

modulators (SERMs), anti-progesterones, estrogen receptor down-regulators (ERDs), estrogen

receptor antagonists, leutinizing hormone-releasing hormone agonists, anti-androgens, aromatase

inhibitors, EGFR inhibitors, VEGF inhibitors, anti-sense oligonucleotides that that inhibit

expression of genes implicated in abnormal cell proliferation or tumor growth.

Chemotherapeutic agents useful in the treatment methods of the present invention include

cytostatic and/or cytotoxic agents.

"Chothia" means an antibody numbering system described in Al-Lazikani et al.,

JMB 273:927-948 (1997).

"Conservatively modified variants" or "conservative substitution" refers to

substitutions of amino acids in a protein with other amino acids having similar characteristics

(e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity,

etc.), such that the changes can frequently be made without altering the biological activity or

other desired property of the protein, such as antigen affinity and/or specificity. Those of skill in

the art recognize that, in general, single amino acid substitutions in non-essential regions of a

polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987)

Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In

addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt

biological activity. Exemplary conservative substitutions are set forth in Table 1.

Table 1. Exemplary Conservative Amino Acid Substitutions Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys; His

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WO wo 2019/160751 PCT/US2019/017177

Original residue Conservative substitution Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val

Lys (K) Arg; His Met (M) Leu; Ile; Tyr

Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

"Consists essentially of," and variations such as "consist essentially of" or

"consisting essentially of," as used throughout the specification and claims, indicate the inclusion

of any recited elements or group of elements, and the optional inclusion of other elements, of

similar or different nature than the recited elements, that do not materially change the basic or

novel properties of the specified dosage regimen, method, or composition. As a non-limiting

example, a PD-1 antigen binding fragment that consists essentially of a recited amino acid

sequence may also include one or more amino acids, including substitutions of one or more

amino acid residues, which do not materially affect the properties of the binding compound.

"Comprising" or variations such as "comprise", "comprises" or "comprised of"

are used throughout the specification and claims in an inclusive sense, i.e., to specify the

presence of the stated features but not to preclude the presence or addition of further features that

may materially enhance the operation or utility of any of the embodiments of the invention,

unless the context requires otherwise due to express language or necessary implication.

"Diagnostic anti-PD-L monoclonal antibody" means a mAb which specifically

binds to the mature form of the designated PD-L (PD-L1 or PD-L2) that is expressed on the

surface of certain mammalian cells. A mature PD-L lacks the presecretory leader sequence, also

referred to as leader peptide The terms "PD-L" and "mature PD-L" are used interchangeably

herein, and shall be understood to mean the same molecule unless otherwise indicated or readily

apparent from the context.

As used herein, a diagnostic anti-human PD-L1 mAb or an anti-hPD-L1 mAb

refers to a monoclonal antibody that specifically binds to mature human PD-L1. A mature human

PD-L1 molecule consists of amino acids 19-290 of the following sequence: wo WO 2019/160751 PCT/US2019/017177

RIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKOLDLAALIVYWEMEDKNI IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFN. TSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALT FIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO:17). Specific examples of diagnostic anti-human PD-L1 mAbs useful as diagnostic

mAbs for immunohistochemistry (IHC) detection of PD-L1 expression in formalin-fixed,

paraffin-embedded (FFPE) tumor tissue sections are antibody 20C3 and antibody 22C3, which

are described in WO 2014/100079. These antibodies comprise the light chain and heavy chain

variable region amino acid sequences shown in Table 2 below:

Table 2. Monoclonal Antibodies 20C3 and 22C3

20C3 Light Chain Mature Variable Region

DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQ KPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA SEQ ID NO:18 VYYCQQSYDVVTFGAGTKLELK

20C3 Heavy Chain Mature Variable Region

QVQVQQSGAELAEPGASVKMSCKASGYIFTSYWMHWLKQRPGG QVQVQQSGAELAEPGASVKMSCKASGYIFTSYWMHWLKQRPGQ GLEWIGYINPSSDYNEYSEKFMDKATLTADKASTTAYMQLISLTS SEQ ID NO:19 EDSAVYYCARSGWLVHGDYYFDYWGQGTTLTVSS

22C3 Light Chain Mature Variable Region

DIVMSQSPSSLAVSAGEKVTMTCKSSQSLLHTSTRKNYLAWYQQ KPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA SEQ ID NO:20 VYYCKQSYDVVTFGAGTKLELK

22C3 Heavy Chain Mature Variable Region

QVHLQQSGAELAKPGASVKMSCKASGYTFTSYWIHWIKQRPGQG QVHLQQSGAELAKPGASVKMSCKASGYTFTSYWIHWIKQRPGQG LEWIGYINPSSGYHEYNQKFIDKATLTADRSSSTAYMHLTSLTSED SEQ ID NO:21

SAVYYCARSGWLIHGDYYFDFWGQGTTLTVSS SAVYYCARSGWLIHGDYYFDFWGQGTTLTVSS Another anti-human PD-L1 mAb that has been reported to be useful for IHC

detection of PD-L1 expression in FFPE tissue sections (Chen, B.J. et al., Clin Cancer Res 19:

3462-3473 (2013)) is a rabbit anti-human PD-L1 mAb publicly available from Sino Biological,

Inc. (Beijing, P.R. China; Catalog number 10084-R015).

"Framework region" or "FR" as used herein means the immunoglobulin variable

regions excluding the CDR regions.

"Isolated antibody" and "isolated antibody fragment" refers to the purification

status and in such context means the named molecule is substantially free of other biological

WO wo 2019/160751 PCT/US2019/017177

molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular

debris and growth media. Generally, the term "isolated" is not intended to refer to a complete

absence of such material or to an absence of water, buffers, or salts, unless they are present in

amounts that substantially interfere with experimental or therapeutic use of the binding

compound as described herein.

"Kabat," as used herein, means an immunoglobulin alignment and numbering

system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest,

5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

"Monoclonal antibody" or "mAb" or "Mab", as used herein, refers to a population

of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population

are identical in amino acid sequence except for possible naturally occurring mutations that may

be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations

typically include a multitude of different antibodies having different amino acid sequences in

their variable domains, particularly their CDRs, which are often specific for different epitopes.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a

substantially homogeneous population of antibodies, and is not to be construed as requiring

production of the antibody by any particular method. For example, the monoclonal antibodies to

be used in accordance with the present invention may be made by the hybridoma method first

described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA

methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated

from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature

352: 624-628 and Marks et al. (1991) J. Mol. Biol, 222: 581-597, for example. See also Presta

(2005) J. Allergy Clin. Immunol. 116:731.

An "anti-PD-1 antibody" useful in the any of the treatment methods, compositions

and uses of the present invention include monoclonal antibodies (mAb), or antigen binding

fragments thereof, which specifically bind to human PD-1. Alternative names or synonyms for

PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1,

B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatment methods, compositions and uses of the present invention in

which a human individual is being treated, the PD-1 antibody or antigen binding fragment

thereof is a PD-1 antagonist that blocks binding of human PD-L1 to human PD-1, or blocks

binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP 054862 and NP_079515, respectively. An anti-PD-1

antibody may be a human antibody, a humanized antibody or a chimeric antibody, and may

include a human constant region. In some embodiments the human constant region is selected

from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred

embodiments, the human constant region is an IgG1 or IgG4 constant region. In some

WO wo 2019/160751 PCT/US2019/017177

embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-

SH, F(ab')2, scFv and Fv fragments.

"PD-L1" or "PD-L2" expression means any detectable level of expression of the

designated PD-L protein on the cell surface or of the designated PD-L mRNA within a cell or

tissue, unless otherwise defined. PD-L protein expression may be detected with a diagnostic PD-

L antibody in an IHC assay of a tumor tissue section or by flow cytometry. Alternatively, PD-L

protein expression by tumor cells may be detected by PET imaging, using a binding agent (e.g.,

antibody fragment, affibody and the like) that specifically binds to the desired PD-L target, e.g.,

PD-L1 or PD-L2. Techniques for detecting and measuring PD-L mRNA expression include RT-

PCR and real-time quantitative RT-PCR.

Several approaches have been described for quantifying PD-L1 protein expression

in IHC assays of tumor tissue sections. See, e.g., Thompson et al., PNAS 101 (49): 17174-17179

(2004); Thompson et al., Cancer Res. 66:3381-3385 (2006); Gadiot et al., Cancer 117:2192-

2201 (2011); Taube et al., Sci Transl Med 4, 127ra37 (2012); and Toplian et al., New Eng. J

Med. 366 (26): 2443-2454 (2012).

One approach employs a simple binary end-point of positive or negative for PD-

L1 expression, with a positive result defined in terms of the percentage of tumor cells that exhibit

histologic evidence of cell-surface membrane staining. A tumor tissue section is counted as

positive for PD-L1 expression is at least 1%, and preferably 5% of total tumor cells.

In another approach, PD-L1 expression in the tumor tissue section is quantified in

the tumor cells as well as in infiltrating immune cells, which predominantly comprise

lymphocytes. The percentage of tumor cells and infiltrating immune cells that exhibit membrane

staining are separately quantified as 5%, 5 to 9%, and then in 10% increments up to 100%. For

tumor cells, PD-L1 expression is counted as negative if the score is < 5% score and positive if

the score is 5%. PD-L1 expression in the immune infiltrate is reported as a semi-quantitative

measurement called the adjusted inflammation score (AIS), which is determined by multiplying

the percent of membrane staining cells by the intensity of the infiltrate, which is graded as none

(0), mild (score of 1, rare lymphocytes), moderate (score of 2, focal infiltration of tumor by

lymphohisticcytic aggregates), or severe (score of 3, diffuse infiltration). A tumor tissue section

is counted as positive for PD-L1 expression by immune infiltrates if the AIS is > 5.

A tissue section from a tumor that has been stained by IHC with a diagnostic PD-

L1 antibody may also be scored for PD-L1 protein expression by assessing PD-L1 expression in

both the tumor cells and infiltrating immune cells in the tissue section using a scoring process.

See WO 2014/165422. One PD-L1 scoring process comprises examining each tumor nest in the

tissue section for staining, and assigning to the tissue section one or both of a modified H score

(MHS) and a modified proportion score (MPS). To assign the MHS, four separate percentages

are estimated across all of the viable tumor cells and stained mononuclear inflammatory cells in

all of the examined tumor nests: (a) cells that have no staining (intensity = 0), (b) weak staining

(intensity =1+), (c) moderate staining (intensity =2+) and (d) strong staining (intensity =3+). A cell must have at least partial membrane staining to be included in the weak, moderate or strong staining percentages. The estimated percentages, the sum of which is 100%, are then input into the formula of 1 X (percent of weak staining cells) + 2 X (percent of moderate staining cells) + 3 X

(percent of strong staining cells), and the result is assigned to the tissue section as the MHS. The

MPS is assigned by estimating, across all of the viable tumor cells and stained mononuclear

inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least

partial membrane staining of any intensity, and the resulting percentage is assigned to the tissue

section as the MPS. In some embodiments, the tumor is designated as positive for PD-L1

expression if the MHS or the MPS is positive.

Another method for scoring/quantifying PD-L1 expression in a tumor is the

"combined positive score" or "CPS," which refers to an algorithm for determining a PD-L1

expression score from a tumor sample of a patient. The CPS is useful in selecting patients for

treatment with particular treatment regimens including methods of treatment comprising

administration of an anti-PD-1 antibody in which expression of PD-L1 is associated with a

higher response rate in a particular patient population relative to same patient population that

does not express PD-L1. The CPS is determined by determining the number of viable PD-L1

positive tumor cells, the number of viable PD-L1 negative tumor cells, and the number of viable

PD-L1 positive mononuclear inflammatory cells (MIC) in a tumor tissue from a patient having a

tumor and calculating the CPS using the following formula:

(# PD-L1 positive tumor cells) + (# PD-L1 positive MIC) X 100% (# PD-L1 positive tumor cells) + (PD-L1 negative tumor cells).

In particular embodiments, the PD-L1 expression scoring method used is the

"lymphoma proportion score." Lymphoma is characterized by a homogeneous population of

confluent cells which efface the architecture of the lymph node or the architecture of metastatic

site. The "LPS" or "lymphoma proportion score" is the percentage of this population of cells

which express PD-L1. When determining the LPS, no attempt is made to distinguish the truly

neoplastic cells from the reactive cells. PD-L1 expression is characterized by partial or

complete membrane staining at any intensity.

Yet another scoring method for PD-L1 expression is the "TPS" or "tumor

proportion score," which is the percentage of tumor cells expressing PD-L1 on the cell

membrane. TPS typically includes the percentage of neoplastic cells expressing PD-L1 at any

intensity (weak, moderate, or strong), which can be determining using an immunohistochemical

assay using a diagnostic anti-human PD-L1 mAb, e.g. antibody 20C3 and antibody 22C3,

described, supra. Cells are considered to express PD-L1 if membrane staining is present,

including cells with partial membrane staining.

The level of PD-L mRNA expression may be compared to the mRNA expression levels of one or more reference genes that are frequently used in quantitative RT-PCR, such as

ubiquitin C.

In some embodiments, a level of PD-L1 expression (protein and/or mRNA) by

malignant cells and/or by infiltrating immune cells within a tumor is determined to be

"overexpressed" or "elevated" based on comparison with the level of PD-L1 expression (protein

and/ or mRNA) by an appropriate control. For example, a control PD-L1 protein or mRNA

expression level may be the level quantified in nonmalignant cells of the same type or in a

section from a matched normal tissue. In some preferred embodiments, PD-L1 expression in a

tumor sample is determined to be elevated if PD-L1 protein (and/or PD-L1 mRNA) in the sample

is at least 10%, 20%, or 30% greater than in the control.

"Tissue section" refers to a single part or piece of a tissue sample, e.g., a thin slice

of tissue cut from a sample of a normal tissue or of a tumor.

"Tumor" as it applies to a subject diagnosed with, or suspected of having, a cancer

refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes

primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue

that usually does not contain cysts or liquid areas. Different types of solid tumors are named for

the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and

lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National

Cancer Institute, Dictionary of Cancer Terms).

"Variable regions" or "V region" as used herein means the segment of IgG chains

which is variable in sequence between different antibodies. It extends to Kabat residue 109 in

the light chain and 113 in the heavy chain.

"RECIST 1.1 Response Criteria" as used herein means the definitions set forth in

Eisenhauer, E.A. et al., Eur. J. Cancer 45:228-247 (2009) for target lesions or non-target lesions,

as appropriate based on the context in which response is being measured.

II. PD-1 Antibodies and Antigen Binding Fragments Useful in the Invention

Examples of mAbs that bind to human PD-1, useful in the treatment methods,

compositions, and uses of the invention, are described in US 7,521,051, US 8,008,449, and US

8,354,509. Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in the treatment

methods, compositions, and uses of the present invention include: pembrolizumab (formerly

known as MK-3475, SCH 900475 and lambrolizumab), a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) and which

comprises the heavy and light chain amino acid sequences shown in FIGURE 1, and the

humanized antibodies h409A11, h409A16 and h409A17, which are described in WO

2008/156712 and in Table 3.

In some embodiments of the treatment methods, compositions, kits and uses of

the present invention, the anti-PD-1 antibody, or antigen binding fragment thereof, comprises: (a)

light chain CDRs comprising a sequence of amino acids as set forth in SEQ ID NOs: 1, 2 and 3

and heavy chain CDRs comprising a sequence of amino acids as set forth in SEQ ID NOs: 6, 7

and 8; or (b) light chain CDRs comprising a sequence of amino acids as set forth in SEQ ID

WO wo 2019/160751 PCT/US2019/017177

NOs: 11, 12 and 13 and heavy chain CDRs comprising a sequence of amino acids as set forth in

SEQ ID NOs: 14, 15 and 16. In some embodiments of the invention, the anti-PD-1 antibody or

antigen binding fragment thereof is a human antibody. In other embodiments, the anti-PD-1

antibody or antigen binding fragment thereof is a humanized antibody. In other embodiments,

the anti-PD-1 antibody or antigen binding fragment thereof is a chimeric antibody. In specific

embodiments, the anti-PD-1 antibody or antigen binding fragment thereof is a monoclonal

antibody.

In other embodiments of the treatment methods, compositions, kits and uses of the

present invention, the PD-1 antibody, or antigen binding fragment thereof, specifically binds to

human PD-1 and comprises (a) a heavy chain variable region comprising an amino acid sequence

as set forth in SEQ ID NO:9, or a variant thereof, and (b) a light chain variable region comprising

an amino acid sequence selected from the group consisting of SEQ ID NO:4 or a variant thereof;

SEQ ID NO:22 or a variant thereof; and SEQ ID NO:23 or a variant thereof.

A variant of a heavy chain variable region sequence or full-length heavy chain

sequence is identical to the reference sequence except having up to 17 conservative amino acid

substitutions in the framework region (i.e., outside of the CDRs), and preferably has less than

ten, nine, eight, seven, six or five conservative amino acid substitutions in the framework region.

A variant of a light chain variable region sequence or full-length light chain sequence is identical

to the reference sequence except having up to five conservative amino acid substitutions in the

framework region (i.e., outside of the CDRs), and preferably has less than four, three or two

conservative amino acid substitution in the framework region.

In another embodiment of the treatment methods, compositions, kits and uses of

the present invention, the PD-1 antibody or antigen-binding fragment thereof is a monoclonal

antibody which specifically binds to human PD-1 and comprises (a) a heavy chain comprising or

consisting of a sequence of amino acids as set forth in SEQ ID NO: 10, or a variant thereof; and

(b) a light chain comprising or consisting of a sequence of amino acids as set forth in SEQ ID

NO:5, or a variant thereof; SEQ ID NO:24, or a variant thereof; or SEQ ID NO:25, or a variant

thereof.

In yet another embodiment of the treatment methods, compositions and uses of

the invention, the PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody

which specifically binds to human PD-1 and comprises (a) a heavy chain comprising or

consisting of a sequence of amino acids as set forth in SEQ ID NO: 10 and (b) a light chain

comprising or consisting of a sequence of amino acids as set forth in SEQ ID NO:5.

Table 3 below provides a list of the amino acid sequences of exemplary anti-PD-1

mAbs for use in the treatment methods, compositions, kits and uses of the present invention.

WO wo 2019/160751 PCT/US2019/017177

Table 3. Exemplary anti-human PD-1 antibodies

A. Comprises light and heavy chain CDRs of hPD-1.09A in WO2008/156712 (light and

heavy chain CDRs of pembrolizumab)

CDRL1 SEQ ID NO:1

CDRL2 SEQ ID NO:2

CDRL3 SEQ ID NO:3

CDRH1 SEQ ID NO:6

CDRH2 SEQ ID NO:7

CDRH3 SEQ ID NO:8 B. Comprises light and heavy chain CDRs of hPD-1.08A in WO2008/156712

CDRL1 SEQ ID NO:11

CDRL2 SEQ ID NO:12

CDRL3 SEQ ID NO:13

CDRH1 SEQ ID NO:14

CDRH2 SEQ ID NO:15

CDRH3 SEQ ID NO:16 C. Comprises the mature h 109A heavy chain variable region (VH) and one of the mature

K09A light chain variable (VL) regions in WO 2008/156712

Heavy chain VH SEQ ID NO:9 (VH of pembrolizumab)

SEQ ID NO:4 (VL of pembrolizumab) or SEQ ID NO:22 or SEQ Light chain VL ID NO:23 D. Comprises the mature 409 heavy chain and one of the mature K09A light chains in

WO 2008/156712 Heavy chain SEQ ID NO:10 (heavy chain of pembrolizumab) SEQ ID NO:5 (light chain of pembrolizumab) or SEQ ID NO:24 or Light chain SEQ ID NO:25

III. Methods and Uses of the Invention

The invention provides a method of treating cancer in a human patient comprising

administering about 400 mg of an anti-PD-1 antibody, or antigen-binding fragment thereof, to the

patient once every about six weeks, wherein the anti-PD-1 antibody or antigen binding fragment

thereof comprises: (a) light chain complementarity determining regions (CDRs) comprising a

sequence of amino acids as set forth in SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs

comprising a sequence of amino acids as set forth in SEQ ID NOs: 6, 7 and 8; or (b) light chain

CDRs comprising a sequence of amino acids as set forth in SEQ ID NOs: 11, 12 and 13 and

heavy chain CDRs comprising a sequence of amino acids as set forth in SEQ ID NOs: 14, 15 and

16. In particular embodiments of the invention, the anti-PD-1 antibody, or antigen-binding

fragment thereof, is pembrolizumab.

In some embodiments of the invention, the anti-PD-1 antibody, or antigen binding

fragment thereof, is administered to the patient about once every six weeks for 12 weeks or more.

In other embodiments, the anti-PD-1 antibody, or antigen binding fragment thereof is

administered to the patient once every six weeks for 18 weeks or more, 24 weeks or more, 30

weeks or more, 36 weeks or more, 42 weeks or more, 48 weeks or more, 54 weeks or more, 60

weeks or more, 66 weeks or more, 72 weeks or more, 78 weeks or more, 84 weeks or more, or 90

weeks or more.

In a first embodiment (Embodiment E1), the invention comprises a method of

treating cancer in a human patient comprising administering 400 mg of an anti-PD-1 antibody

(e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks.

In a second embodiment (Embodiment E2), the invention comprises a method of

treating unresectable or metastatic melanoma in a human patient comprising administering 400

mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the

patient once every approximately six weeks.

In a third embodiment (Embodiment E3), the invention comprises a method of

treating metastatic non-small cell lung cancer (NSCLC) in a human patient comprising

administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding

fragment thereof, to the patient once every approximately six weeks.

In a sub-embodiment of Embodiment E3 (Embodiment E3-A), the patient has a

tumor with high PD-L1 expression [(Tumor Proportion Score (TPS) >50%)] and was not

previously treated with platinum-containing chemotherapy.

In a further sub-embodiment of Embodiment E3 (Embodiment E3-B), the patient

has a tumor with PD-L1 expression (TPS >1%) and was previously treated with platinum-

containing chemotherapy. In specific embodiments of Embodiment E3-B, the patient had

disease progression on or after receiving platinum-containing chemotherapy.

In another sub-embodiment of Embodiment E3 (Embodiment E3-C), the patient

has a tumor with PD-L1 expression (TPS 1%) and was not previously treated with platinum-

containing chemotherapy.

In yet another sub-embodiment of Embodiment E3 (Embodiment E3-D), the

patient's tumor is not tested for PD-L1 expression. In this embodiment, the patient is treated

with the anti-PD-1 antibody, or antigen binding fragment thereof, regardless of PD-L1

expression. In specific embodiments, the patient was not previously treated with platinum-

containing chemotherapy.

In certain embodiments of Embodiment E3 (including Embodiment E3-A, E3-B,

and E3-C), the PD-L1 TPS is determined by an FDA-approved test.

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In certain embodiments of Embodiment E3 (including Embodiment E3-A, E3-B,

E3-C and E3-D), the patient's tumor has no EGFR or ALK genomic aberrations.

In certain embodiments of Embodiment E3 (including Embodiment E3-A, E3-B,

E3-C and E3-D), the patient's tumor has an EGFR or ALK genomic aberration and had disease

progression on or after receiving treatment for the EGFR or ALK aberration(s) prior to receiving

the anti-PD-1 antibody, or antigen binding fragment thereof.

In a fourth embodiment (Embodiment E4), the invention comprises a method of

treating metastatic non-small cell lung cancer (NSCLC) in a human patient comprising: (1)

administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding

fragment thereof, to the patient once every approximately six weeks, and (2) administering

pemetrexed and carboplatin to the patient. In sub-embodiments of Embodiment E4, the patient

was not previously treated with an anti-cancer therapeutic prior to starting the combination

treatment regimen with the anti-PD-1 antibody, or antigen binding fragment thereof, pemetrexed

and carboplatin.

In certain embodiments of Embodiment E3 and E4 (including sub-embodiments

thereof), the patient has nonsquamous non-small cell lung cancer.

In sub-embodiments of Embodiment E4, pemetrexed is administered to the patient in an amount of 500 mg/m².

In sub-embodiments of Embodiment E4, pemetrexed is administered to the patient

via intravenous infusion every 21 days. In specific embodiments, the infusion time is about 10

minutes.

In sub-embodiments of Embodiment E4 (Embodiment E4-A), the invention

further comprises administering about 400 ug to about 1000 ug of folic acid to the patient once

per day, beginning about 7 days prior to administering pemetrexed to the patient and continuing

until about 21 days after the patient is administered the last dose of pemetrexed. In certain

embodiments the folic acid is administered orally.

In sub-embodiments of Embodiments E4 and E4-A (Embodiment E4-B), the invention further comprises administering about 1 mg of vitamin B12 to the patient about 1 week

prior to the first administration of pemetrexed and about every three cycles of pemetrexed

administration (i.e., approximately every 9 weeks). In certain embodiments the vitamin B12 is

administered intramuscularly.

In sub-embodiments of Embodiments E4, E4-A and E4-B (Embodiment E4-C), the invention further comprises administering about 4 mg of dexamethasone to the patient twice

a day on the day before, the day of, and the day after pemetrexed administration. In certain

embodiments the dexamethasone is administered orally.

In a fifth embodiment (Embodiment E5), the invention comprises a method of

treating recurrent or metastatic head and neck squamous cell cancer (HNSCC) in a human patient

comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen

binding fragment thereof, to the patient once every approximately six weeks.

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WO wo 2019/160751 PCT/US2019/017177

In sub-embodiments of Embodiment E5, the patient was previously treated with

platinum-containing chemotherapy. In certain embodiments, the patient had disease progression

on or after platinum-containing chemotherapy.

In a sixth embodiment (Embodiment E6), the invention comprises a method of

treating refractory classical Hodgkin lymphoma (cHL) in a human patient comprising

administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding

fragment thereof, to the patient once every approximately six weeks.

In a seventh embodiment (Embodiment E7), the invention comprises a method of

treating classical Hodgkin lymphoma (cHL) in a human patient comprising administering 400 mg

of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the

patient once every approximately six weeks, wherein the patient has relapsed after (a) one or

more lines of therapy for cHL, (b) 2 or more lines of therapy for cHL, or (c) 3 or more lines of

therapy for cHL.

In sub-embodiments of Embodiments E6 and E7, the patient is an adult patient.

In alternative sub-embodiments of Embodiments E6 and E7, the patient is a

pediatric patient.

In an eighth embodiment (Embodiment E8), the invention comprises a method of

treating locally advanced or metastatic urothelial carcinoma in a human patient comprising

administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding

fragment thereof, to the patient once every approximately six weeks.

In sub-embodiments of Embodiment E8, the patient is not eligible for cisplatin-

containing chemotherapy.

In sub-embodiments of Embodiment E8, the patient had disease progression during

or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant

treatment with platinum-containing chemotherapy.

In sub-embodiments of Embodiment E8, the patient's tumor expresses PD-L1

(CPS >10).

In a ninth embodiment (Embodiment E9), the invention comprises a method of

treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair

(MMR) deficient solid tumors in a human patient comprising administering 400 mg of an anti-

PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once

every approximately six weeks.

In a sub-embodiment of Embodiment E9, the patient had disease progression

following prior anti-cancer treatment.

In a tenth embodiment (Embodiment E10), the invention comprises a method of

treating unresectable or metastatic, MSI-H or MMR deficient colorectal cancer in a human

patient comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or

antigen binding fragment thereof, to the patient once every approximately six weeks.

In a sub-embodiment of Embodiment E10, the patient had disease progression

following prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

In an eleventh embodiment (Embodiment E11), the invention comprises a method

of treating recurrent locally advanced or metastatic gastric cancer in a human patient comprising

administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding

fragment thereof, to the patient once every approximately six weeks.

In a twelfth embodiment (Embodiment E12), the invention comprises a method of

treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma in a

human patient comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab),

or antigen binding fragment thereof, to the patient once every approximately six weeks.

In sub-embodiments of Embodiments E11 and E12, the patient's tumor expresses

PD-L1 [Combined Positive Score (CPS) 1].

In sub-embodiments of Embodiments E11 and E12, the patient had disease

progression on or after one or more prior lines of therapy. In specific embodiments, the prior

lines of therapy include fluoropyrimidine and platinum-containing chemotherapy.

In sub-embodiments of Embodiments E11 and E12, the patient had disease

progression on or after two or more prior lines of therapy including fluoropyrimidine- and

platinum-containing chemotherapy.

In sub-embodiments of Embodiments E11 and E12, the patient had disease

progression on or after one or more prior lines of therapy including HER2/neu-targeted therapy.

In sub-embodiments of Embodiments E11 and E12, the patient had disease

progression on or after two or more prior lines of therapy including HER2/neu-targeted therapy.

In a thirteenth embodiment (Embodiment E13), the invention comprises a method

of treating cancer in a human patient comprising administering 400 mg of an anti-PD-1 antibody

(e.g. pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks, wherein the patient has a cancer selected from the group consisting of:

melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal

cancer, multiple myeloma, hepatocellular cancer, lymphoma, renal cancer, mesothelioma,

ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical

cancer, thyroid cancer, and salivary cancer.

In a fourteenth embodiment (Embodiment E14), the invention comprises a

method of treating cancer in a human patient comprising administering 400 mg of an anti-PD-1

antibody (e.g. pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks, wherein the patient has small-cell lung cancer.

In a fifteenth embodiment (Embodiment E15), the invention comprises a method

of treating non-Hodgkin lymphoma in a human patient comprising administering 400 mg of an

anti-PD-1 antibody (e.g. pembrolizumab), or antigen binding fragment thereof, to the patient

once every approximately six weeks.

WO wo 2019/160751 PCT/US2019/017177

In a sub-embodiment of Embodiment E15, the non-Hodgkin lymphoma is primary

mediastinal large B-cell lymphoma (PMBCL). In some embodiments where the patient has

PMBCL, the patient has refractory PMBCL. In some embodiments, the patient has relapsed after

one or more prior lines of therapy. In some embodiments, the patient has relapsed after two or

more prior lines of therapy. In some embodiments, the patient was not previously treated with

another line of therapy.

In a sixteenth embodiment (Embodiment E16), the invention comprises a method

of treating metastatic squamous NSCLC in a human patient comprising: (1) administering 400

mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the

patient once every approximately six weeks, and (2) administering (i) carboplatin and paclitaxel,

or (ii) carboplatin and nab-paclitaxel to the patient.

In a seventeenth embodiment (Embodiment E17), the invention comprises a

method of treating Merkel cell carcinoma (MCC) in a human patient comprising administering

400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to

the patient once every approximately six weeks. In particular sub-embodiments of Embodiment

E17, the cancer is recurrent, locally advanced MCC. In particular sub-embodiments of

Embodiment E17, the cancer is metastatic MCC.

In sub-embodiments of Embodiment E17, the patient is an adult patient. In

alternative sub-embodiments of Embodiment E17, the patient is a pediatric patient.

In a eighteenth embodiment (Embodiment E18), the invention comprises a method

for adjuvant therapy of melanoma in a human patient comprising administering 400 mg of an

anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to a patient once

every approximately six weeks, wherein the patient has previously had one or more melanoma

lesions resected. In sub-embodiments of Embodiment E18, the method comprises treating

resected high-risk stage III melanoma.

In a nineteenth embodiment (Embodiment E19), the invention comprises a method

of treating hepatocellular carcinoma (HCC) in a human patient comprising administering 400 mg

of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the

patient once every approximately six weeks. In some embodiments of Embodiment E19, the

patient was previously treated with sorafenib.

In a twentieth embodiment (Embodiment E20), the invention comprises a method

of treating renal cell carcinoma (RCC) in a human patient comprising administering 400 mg of

an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient

once every approximately six weeks.

In sub-embodiments, of Embodiment E20, the cancer is advanced clear cell RCC.

In sub-embodiments of Embodiment E20, the patient has advanced or metastatic

renal cell carcinoma (RCC).

In sub-embodiments, of Embodiment E20 (Embodiment E20A), the patient is

further treated with axitinib. In sub-embodiments of the invention, axitinib is taken orally.

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In particular embodiments of Embodiment E20A, 5 mg axitinib is taken by the

patient approximately every 12 hours or twice a day.

In alternative embodiments of Embodiment E20A, the axitinib dosage is 2.5 mg, 3

mg, 7 mg, or 10 mg twice daily.

In a twenty-first embodiment (Embodiment E21), the invention comprises a

method of treating breast cancer in a human patient comprising administering 400 mg of an anti-

PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once

every approximately six weeks.

In a sub-embodiment of Embodiment E21, the breast cancer is triple negative

breast cancer.

In a sub-embodiment of Embodiment E21, the breast cancer is ER+/HER2- breast

cancer.

In a twenty-second embodiment (Embodiment E22), the invention comprises a

method of treating nasopharyngeal cancer in a human patient comprising administering 400 mg

of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the

patient once every approximately six weeks.

In a twenty-third embodiment (Embodiment E23), the invention comprises a

method of treating thyroid cancer in a human patient comprising administering 400 mg of an

anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient

once every approximately six weeks.

In a twenty-fourth embodiment (Embodiment E24), the invention comprises a

method of treating salivary cancer in a human patient comprising administering 400 mg of an

anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient

once every approximately six weeks.

In a twenty-fifth embodiment (Embodiment E25), the invention comprises a

method of treating cancer in a human patient comprising administering 400 mg of an anti-PD-1

antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks, wherein the cancer is selected from the group consisting of: melanoma,

non-small cell lung cancer, relapsed or refractory classical Hodgkin lymphoma, primary

mediastinal large B-cell lymphoma, head and neck squamous cell cancer, urothelial carcinoma,

esophageal cancer, gastric cancer, cervical cancer, PMBCL, MSI-H cancer, hepatocellular

carcinoma, and Merkel cell carcinoma.

In a twenty-sixth embodiment (Embodiment E26), the invention comprises a

method of treating cancer in a human patient comprising administering 400 mg of an anti-PD-1

antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks, wherein the cancer is a Heme malignancy.

In a sub-embodiment of Embodiment E26, the heme malignancy is selected from

the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),

chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T- cell/histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (MCL-

1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), and small lymphocytic

lymphoma (SLL). In a twenty-seventh embodiment (Embodiment E27), the invention comprises a

method of treating cancer in a human patient comprising administering 400 mg of an anti-PD-1

antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient once every

approximately six weeks, wherein the patient has a tumor with a high mutational burden.

In specific embodiments, a high mutational burden is at least about 10 mutations

per megabase of genome examined, at least about 11 mutations per megabase of genome

examined, at least about 12 mutations per megabase of genome examined, or at least about 13

mutations per megabase of genome examined.

In a twenty-eighth embodiment (Embodiment E28), the invention comprises a

method of treating esophageal cancer in a human patient comprising administering 400 mg of an

anti-PD-1 antibody (e.g., pembrolizumab), or antigen binding fragment thereof, to the patient

once every approximately six weeks.

In sub-embodiments of Embodiment E28, the patient progressed with one previous

line of standard therapy prior to receiving the anti-PD-1 antibody, or antigen binding fragment

thereof. In a further embodiment, the patient progressed with one or more lines of standard

therapy prior to receiving the anti-PD-1 antibody, or antigen binding fragment thereof. In

another embodiment, the patient progressed with two or more lines of standard therapy prior to

receiving the anti-PD-1 antibody, or antigen binding fragment thereof. In particular

embodiments, the standard therapy includes one or more of: paclitaxel, docetaxel, or irinotecan.

In sub-embodiments of Embodiment E28, the patient has advanced or metastatic

adenocarcinoma or squamous cell carcinoma of the esophagus.

In sub-embodiments of Embodiment E28, the patient has advanced or metastatic

Siewert type I adenocarcinoma of the esophagogastric junction.

In sub-embodiments of Embodiment E28, the patient's tumor expresses PD-L1

(Combined Positive Score [CPS] >10).

In a twenty-ninth embodiment (Embodiment E29), the invention comprises a

method of treating high-risk non-muscle invasive bladder cancer (NMIBC) in a human patient

comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab), or antigen

binding fragment thereof, to the patient once every approximately six weeks. In some

embodiments, the patient has NMIBC with carcinoma in situ (CIS) or CIS plus papillary disease.

In a sub-embodiment of Embodiment E29, the patient was previously treated with

standard therapy prior to being treated with the anti-PD-1 antibody, or antigen binding fragment

thereof. In some embodiments, the prior therapy is Bacillus Calmette-Guérin (BCG) therapy. In

WO wo 2019/160751 PCT/US2019/017177

particular embodiments, the patient did not respond to BCG therapy. In some embodiments, the

patient was ineligible for radical cystectomy or chose not to undergo radical cystectomy.

In any of the methods of the invention described above (including Embodiments

E1-E29), the PD-1 antibody or antigen binding fragment is any of the antibodies or antigen-

binding fragments described in Section II of the Detailed Description of the Invention "PD-1

Antibodies and Antigen Binding Fragments Useful in the Invention" herein. In some

embodiments, the anti-PD-1 antibody is pembrolizumab or an antigen-binding fragment thereof,

or an antibody which cross competes with pembrolizumab for binding to human PD-1. In some

embodiments, the anti-PD-1 antibody is a variant of pembrolizumab; i.e. an antibody or antigen-

binding fragment having light chain CDRs comprising a sequence of amino acids as set forth in

SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs comprising a sequence of amino acids as set

forth in SEQ ID NOs: 6, 7 and 8.

In any of the methods of the invention described above (including Embodiments

E1-E29), the PD-1 antibody or antigen binding fragment is administered to the patient once

every approximately six weeks. In particular embodiments, the PD-1 antibody or antigen

binding fragment is administered to the patient every six weeks, every six weeks +5 days, +4

days, 3 days, =2 days or +1 day.

In embodiments of any of the methods of treatment herein, a patient is

administered an intravenous (IV) infusion of a medicament comprising any of the anti-PD-1

antibodies or antigen-binding fragments described herein.

In alternative embodiments of any of the methods of treatment herein, the patient

is administered (e.g., by a clinician) or administers any of the anti-PD-1 antibodies or antigen-

binding fragments subcutaneously.

In any of the methods described herein, including Embodiment E1-E29, and sub-

embodiments thereof, the method may further comprise one or more "additional therapeutic

agents" (as used herein, "additional therapeutic agent" refers to an additional agent relative to the

anti-PD-1 antibody or antigen-binding fragment thereof). The additional therapeutic agent may

be, e.g., a chemotherapeutic other than an anti-PD-1 antibody, a biotherapeutic agent (including

but not limited to antibodies to CTLA4, VEGF, EGFR, Her2/neu, VEGF receptors, other growth

factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), an immunogenic agent (for

example, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic

cells pulsed with tumor derived antigen or nucleic acids, immune stimulating cytokines (for

example, IL-2, IFNa2, GM-CSF), and cells transfected with genes encoding immune stimulating

cytokines such as but not limited to GM-CSF).

As noted above, in some embodiments of the methods of the invention, the

method further comprises administering an additional therapeutic agent. In particular

embodiments, the additional therapeutic agent is an anti-CTLA4 antibody or antigen binding

fragment thereof, an anti-LAG3 antibody or antigen binding fragment thereof, an anti-GITR

antibody, or antigen binding fragment thereof, an anti-TIGIT antibody, or antigen binding wo WO 2019/160751 PCT/US2019/017177 fragment thereof, an anti-CD27 antibody or antigen binding fragment thereof, an anti-ILT3 antibody, or antigen binding fragment thereof, or an anti-ILT4 antibody, or antigen binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease viral vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In another embodiment, the additional therapeutic agent is navarixin. In a further embodiment, the additional therapeutic agent is vicriviroc.

In a further embodiment, the additional therapeutic agent is an oncolytic virus. In

one embodiment, the additional therapeutic agent is Coxsackievirus or CVA21. In one

embodiment, the additional therapeutic agent is CAVATAKTM

In yet another embodiment, the additional therapeutic agent is a STING agonist.

In a further embodiment, the additional therapeutic agent is an IL-27 antagonist. In one

embodiment, the additional therapeutic agent is a PARP inhibitor. In one embodiment, the

additional therapeutic agent is a multi-kinase inhibitor. In one embodiment, the additional

therapeutic agent is a MEK inhibitor. In one embodiment, the additional therapeutic agent is a 4-

1BB agonist.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa

and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan;

aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and

methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,

triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and

bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin;

callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues);

cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin

(including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a

sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,

cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide

hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil

mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,

ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially

calicheamicin gammalI and calicheamicin phill, see, e.g., Agnew, Chem. Intl. Ed. Engl.,

33:183-186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an

esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne

antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,

cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,

daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-

doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),

epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,

mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,

quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;

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WO wo 2019/160751 PCT/US2019/017177

anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as

denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-

mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-

azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;

androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane,

testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher

such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;

amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;

elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;

lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone;

mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-

ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;

triaziquone; 2, ,2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A,

roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol;

mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;

taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;

mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine;

platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone;

teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase

inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid;

capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors

such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for

example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,

LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme

aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-

imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole,

letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide,

leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the

above.

In some embodiments which comprise a step of administering an additional

therapeutic agent (i.e., in addition to the PD-1 antibody (e.g., pembrolizumab) or antigen-binding

fragment thereof), the additional therapeutic agent in the combination therapy may be

administered using the same dosage regimen (dose, frequency and duration of treatment) that is

typically employed when the agent is used as monotherapy for treating the same cancer. In other

embodiments, the patient receives a lower total amount of the additional therapeutic agent in the

combination therapy than when that agent is used as monotherapy, e.g., smaller doses, less

frequent doses, and/or shorter treatment duration.

The additional therapeutic agent in a combination therapy can be administered

orally, intratumorally, or parenterally, including the intravenous, intramuscular, intraperitoneal,

subcutaneous, rectal, topical, and transdermal routes of administration. For example, the

combination treatment may comprise an anti-PD-1 antibody or antigen binding fragment thereof,

and an anti-CTLA antibody or antigen binding fragment thereof, both of which may be

administered intravenously or subcutaneously, as well as a chemotherapeutic agent, which may

be administered orally.

A combination therapy of the invention may be used prior to or following surgery

to remove a tumor and may be used prior to, during or after radiation therapy. A combination

therapy of the invention may also be used when a patient's tumor is non-resectable.

In some embodiments, a combination therapy of the invention is administered to a

patient who has not been previously treated with a biotherapeutic or chemotherapeutic agent, i.e.,

is treatment-naîve. In other embodiments, the combination therapy is administered to a patient

who failed to achieve a sustained response after prior therapy with a biotherapeutic or

chemotherapeutic agent, i.e., is treatment-experienced.

A combination therapy of the invention may be used to treat a tumor that is large

enough to be found by palpation or by imaging techniques well known in the art, such as MRI,

ultrasound, or CAT scan. In some embodiments, a combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm 3, 300 mm³ 3 400

mm³ 500 mm³ 750 mm³, or up to 1000 mm³.

In some embodiments, a combination therapy of the invention is administered to a

human patient who has a cancer that expresses PD-L1. In some embodiments, PD-L1 expression

is detected using a diagnostic anti-human PD-L1 antibody, or antigen binding fragment thereof,

in an IHC assay on an FFPE or frozen tissue section of a tumor sample removed from the patient.

A patient's physician may order a diagnostic test to determine PD-L1 expression in a tumor

tissue sample removed from the patient prior to initiation of treatment with the anti-PD-1

antibody, or antigen-binding fragment thereof, but it is envisioned that the physician could order

the first or subsequent diagnostic tests at any time after initiation of treatment, such as for

example after completion of a treatment cycle.

Selecting a dosage of the additional therapeutic agent depends on several factors,

including the serum or tissue turnover rate of the entity, the level of symptoms, the

immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the

individual being treated. The dosage of the additional therapeutic agent should be an amount that

provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency

of each additional therapeutic agent (e.g. biotherapeutic or chemotherapeutic agent) will depend

in part on the particular therapeutic agent, the severity of the cancer being treated, and patient

characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small

molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.

Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis,

WO wo 2019/160751 PCT/US2019/017177

Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy

in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med.

348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001)

New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619;

Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med.

343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed);

Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002).

Determination of the appropriate dosage regimen may be made by the clinician, e.g., using

parameters or factors known or suspected in the art to affect treatment or predicted to affect

treatment, and will depend, for example, the patient's clinical history (e.g., previous therapy), the

type and stage of the cancer to be treated and biomarkers of response to one or more of the

therapeutic agents in the combination therapy.

IV. Compositions and Kits

The invention also relates to compositions comprising a dosage of an anti-PD-1

antibody (e.g., pembrolizumab) or antigen binding fragment thereof and a pharmaceutically

acceptable carrier or excipient, wherein the dosage is about 400 mg. The anti-PD-1 antibody

may be produced, for example, in CHO cells using conventional cell culture and

recovery/purification technologies.

In embodiments of the invention, the composition further comprises histidine

buffer at about pH 5.0 to pH 6.0. In particular embodiments, the histidine is present in a concentration of about 10 mM.

In embodiments of the invention, the composition further comprises sucrose. In

particular embodiments, the sucrose is present in a concentration of about 70 mg/mL.

In embodiments of the invention, the composition further comprises polysorbate

80. In particular embodiments, the polysorbate 80 is present in a concentration of about 0.2

mg/mL. In some embodiments, the composition comprises 10 mM histidine, pH 5.5, 7%

sucrose, 0.02% polysorbate 80, and 400 mg of the anti-PD-1 antibody or antigen-binding

fragment thereof.

In embodiments of the invention, the composition is liquid.

In alternative embodiments, the composition is lyophilized.

In the compositions of the invention, the anti-PD-1 antibody or antigen binding

fragment thereof can be any of the antibodies and antigen binding fragments described herein,

i.e. described in Section II of the Detailed Description of the Invention "PD-1 Antibodies and

Antigen Binding Fragments Useful in the Invention" (e.g. pembrolizumab).

In some embodiments, a composition comprising an anti-PD-1 antibody as the

PD-1 antagonist may be provided as a liquid formulation or prepared by reconstituting a

lyophilized powder with sterile water for injection prior to use. WO 2012/135408 describes the

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WO wo 2019/160751 PCT/US2019/017177

preparation of liquid and lyophilized medicaments comprising pembrolizumab that are suitable

for use in the present invention.

The invention also relates to a kit for treating a patient with cancer, the kit

comprising: (a) 400 mg of an anti-PD-1 antibody or antigen binding fragment thereof, and (b)

instructions for using the anti-PD-1 antibody or antigen binding fragment thereof in any of the

methods for treating cancer described herein.

In any of the kits of the invention, the PD-1 antibody or antigen binding fragment

can be any of the antibodies or antigen-binding fragments described in Section II of the Detailed

Description of the Invention "PD-1 Antibodies and Antigen Binding Fragments Useful in the

Invention".

The kits of the invention may provide the anti-PD-1 or antigen-binding fragments

thereof in a container and a package insert. The container contains at least one dose (i.e. about

400 mg) of a medicament comprising an anti-PD-1 antibody, or antigen binding fragment

thereof, and the package insert, or label, which comprises instructions for treating a patient with

cancer using the medicament. The container may be comprised of the same or different shape

(e.g., vials, syringes and bottles) and/or material (e.g., plastic or glass). The kit may further

comprise other materials that may be useful in administering the medicaments, such as diluents,

filters, IV bags and lines, needles and syringes. In some preferred embodiments of the kit, the

instructions state that the medicament is intended for use in treating a patient having a tumor,

wherein the tumor expresses PD-L1 by, e.g., an IHC assay. In some embodiments, the tumor has

a tumor proportion score (TPS) of 1% PD-L1. In another embodiment, the tumor has a TPS of

>50% PD-L1. A PD-L1 TPS is the number of tumor cells in a sample expressing PD-L1. In

further embodiments, the tumor has a TPS of>5%PD-L1, >10 PD-L1, >15% PD-L1, >20% PD-

L1, >25% PD-L1, >30% PD-L1, >35% PD-L1, >40% PD-L1, or >45% PD-L1. In another embodiment, the patient's tumor expresses PD-L1 with a CPS of >10%. In another

embodiment, the patient's tumor expresses PD-L1 with a CPS of >5%. In another embodiment,

the patient's tumor expresses PD-L1 with a CPS of >1%.

These and other aspects of the invention, including the exemplary specific

embodiments listed below, will be apparent from the teachings contained herein.

GENERAL METHODS Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu

(1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also

appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley

and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis

WO wo 2019/160751 PCT/US2019/017177

(Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression

(Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation, chromatography,

electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current

Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,

chemical modification, post-translational modification, production of fusion proteins,

glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in

Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current

Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-

16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-

89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).

Production, purification, and fragmentation of polyclonal and monoclonal antibodies are

described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons,

Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for

characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current

Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g.,

Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY;

Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow

and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998)

J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J.

Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter

(1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody libraries displayed on

phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol.

14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics

15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001)

Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory

Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-

399).

Purification of antigen is not necessary for the generation of antibodies. Animals

can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated

from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce

a hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity

13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

32

WO wo 2019/160751 PCT/US2019/017177

Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes,

polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other

purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,

colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol, 146:169-175; Gibellini et al.

(1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;

Everts et al. (2002) J. Immunol, 168:883-889).

Methods for flow cytometry, including fluorescence activated cell sorting

(FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.;

Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons,

Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid

primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are

available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-

Aldrich (2003) Catalogue, St. Louis, MO).

Standard methods of histology of the immune system are described (see, e.g.,

Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag,

New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins,

Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

Software packages and databases for determining, e.g., antigenic fragments, leader sequences,

protein folding, functional domains, glycosylation sites, and sequence alignments, are available

(see, e.g., GenBank, Vector NTIR Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin

Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada);

Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics

Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-

181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.

14:4683-4690).

All publications mentioned herein are incorporated by reference for the purpose of

describing and disclosing methodologies and materials that might be used in connection with the

present invention.

Having described different embodiments of the invention herein with reference to

the accompanying drawings, it is to be understood that the invention is not limited to those

precise embodiments, and that various changes and modifications may be effected therein by one

skilled in the art without departing from the scope or spirit of the invention as defined in the

appended claims.

EXAMPLE 1 A six-weekly (Q6W) dosing schedule for pembrolizumab across multiple tumor types based on

an evaluation using modeling and simulation

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Pembrolizumab, an anti-PD-1 checkpoint inhibitor currently approved for use in

multiple cancer indications, has demonstrated safety and efficacy when administered at a dose of

either 200 mg or 2 mg/kg Q3W. An alternative extended dosing regimen would provide the

benefits of convenience and flexibility to both patients and prescribers. The robust

characterization of pembrolizumab pharmacokinetics (PK) and exposure (concentration)-

response (E-R) relationships for both efficacy and safety allow the use of model-based

approaches to support alternative dosing regimens for pembrolizumab.

The dose for a Q6W schedule of pembrolizumab was selected by matching

exposures with the approved Q3W (200 mg and 2 mg/kg) regimens after PK steady state is

achieved; the efficacy and safety between regimens were bridged based on knowledge of E-R.

PK exposures were simulated up to 24 weeks of dosing, to ensure steady state in all subjects,

using the established population PK model (with time dependent elimination) of pembrolizumab

that adequately described PK across multiple tumor types. Efficacy was bridged using exposure

metrics at steady state, AUCss or time-averaged concentration (Cavg,ss) and trough

concentrations (Cmin,ss), which were compared between regimens. The safety profile of

pembrolizumab at the Q6W schedule was bridged by ensuring that the predicted peak

concentrations at steady state (Cmax,ss) are below those of the maximum clinically administered

and well-tolerated dose of 10 mg/kg Q2W.

The PK of pembrolizumab after administration of 400 mg Q6W is predicted to

follow a similar profile as the PK at the approved 200 mg Q3W and 2 mg/kg Q3W dosing

regimens (see FIG. 4). The exposure metrics as compared between regimens are summarized in

Table 4. The 400 mg Q6W dosing regimen of pembrolizumab was selected based on similar

predicted exposures (Cavg,ss or AUCss, geometric mean (GM) ~1% higher) compared with

those achieved at 200 mg Q3W (see FIG. 3). Less than 1% subjects were predicted to have

Cmin,ss that are lower in comparison with those at 200 mg Q3W and 2 mg/kg Q3W (FIG. 3).

The predicted Cmax,ss for 400 mg Q6W are well below (GM ~65% lower) that achieved with 10

mg/kg Q2W, which has been shown to have acceptable safety across multiple tumor types (see

FIG. 2). Given the similar exposure profiles and the established, flat E-R relationships for

pembrolizumab at clinically tested doses, the clinical outcomes achieved with 400 mg Q6W are

expected to be similar to those with 200 mg Q3W across tumor types.

Based on the modeling and simulation approach used herein, it is expected that a

400 mg Q6W dosing regimen for pembrolizumab would lead to PK exposures that are similar to

the approved 200 mg Q3W and 2 mg/kg dosing regimens. PK simulations demonstrate that in

terms of pembrolizumab exposures - Average concentration over the dosing interval (Cavg) (or

area under the curve [AUC]) at 400 mg Q6W was similar to that at the approved 200 mg Q3W

dose, thus bridging efficacy between dosing regimens. Trough concentrations (Cmin) at 400 mg

Q6W were generally within the range of those achieved with 2 mg/kg or 200 mg Q3W in the

majority (>99%) of patients. Peak concentrations (Cmax) at 400 mg Q6W were well below the

Cmax for the highest clinically tested dose of 10 mg/kg Q2W, supporting that the safety profile

WO wo 2019/160751 PCT/US2019/017177

for 400 mg Q6W should be comparable to the established safety profile of pembrolizumab.

Exposure-response (E-R) for pembrolizumab was demonstrated to be flat across indications, and

os predictions in melanoma and NSCLC demonstrate that efficacy at 400 mg Q6W is expected

to be similar to that at 200 mg or 2 mg/kg Q3W, given the similar exposures; thus 400 mg Q6W

is expected to be efficacious across indications.

Table 4. Summary of Pembrolizumab PK Exposure Metrics for the 400 mg Q6W Dosing Regimen Based on Simulations

Alternative Dosing Regimen Q6W 400 mg Cavg,ss Relative to 200 mg Q3W, 0.7% % difference in GM at steady state

Cmin,ss Relative to 2 mpk Q3W, -12% % difference in GM at steady state

% of patients below lower limit of range for 200 mg and 2 mpk < 1 % Q3W at steady state

Cmax,ss Relative to 10 mpk Q2W, -66 % % difference in GM at steady state

EXAMPLE 2 A Phase 1 Randomized Clinical Study of Pembrolizumab to Evaluate the Safety and Tolerability

of Intravenous Infusion of 400 mg Pembrolizumab Q6W in Participants with Advanced

Melanoma

This study is designed to assess the pharmacokinetics (PK), safety and tolerability

of pembrolizumab when administered every 6 weeks (Q6W). A cohort of 100 participants is

given 400 mg pembrolizumab Q6W. PK, efficacy, and safety data are collected from this cohort

of participants. Male/female participants of at least 18 years of age with advanced melanoma are

enrolled in the study. No stratification based on age, sex, or other characteristics is used in this

study.

Participants receive IV infusion of 400 mg pembrolizumab Q6W from cycles 1 to

18. PK, efficacy, and safety data are collected from these participants. Results provide

preliminary PK, efficacy, and safety data of pembrolizumab when administered Q6W. Based on

the robust understanding of pembrolizumab clinical pharmacology and its well-established E-R

profiles, such a dosing schedule change is expected to produce similar efficacy and safety in all

treatment settings where 200 mg Q3W pembrolizumab is approved (including monotherapy and

WO wo 2019/160751 PCT/US2019/017177

in combination with other agents). Thus, a 400 mg Q6W regimen would have a similar benefit-

risk profile to 200 mg Q3W, as a less frequent dosing regimen in the clinical use of

pembrolizumab based on modeling and simulation analyses (see EXAMPLE 1).

Study Design

The study, which is a randomized, cross-over, multicenter, open-label, safety

study of pembrolizumab in participants with advanced melanoma, is conducted in conformance

with Good Clinical Practices (GCP). This Phase 1 study is conducted in participants with

unresectable or metastatic melanoma. The treatment period continues every 42 days for up to 18

cycles (approximately 2 years). Treatment will continue as long as participants are receiving

benefit from treatment and have not had disease progression or met any criteria for study

withdrawal. In greater detail, the study consists of: (1) A screening period of up to a 28-day

duration to ensure that the participant is eligible for the study and (2) an intervention period of

approximately 104 weeks of treatment with pembrolizumab. Participants receive pembrolizumab

via IV infusion over 30 minutes Q6W for up to 18 cycles, and (3) a follow-up period during

which participants are monitored for AEs for 30 days and serious adverse events (SAEs) for 90

days (30 days if the participant initiates new anticancer therapy). Participants with an ongoing

AE at the time of treatment discontinuation are followed until resolution, stabilization, the event

is otherwise explained, or the participant is lost to follow-up.

Participants who discontinue for reasons other than radiographic disease

progression have post-treatment follow-up imaging for disease status until disease progression is

documented radiographically per RECIST 1.1 and, when clinically appropriate, confirmed by the

site per iRECIST, initiating a non-study cancer treatment, withdrawing consent, becoming lost to

follow-up or the end of the study. All participants are followed by telephone for overall survival

in the Survival follow-up period until death, participant withdrawal of consent, becoming lost to

follow-up or the end of the study.

All participants enrolled into this study will have a diagnosis of advanced

melanoma. The results of this study will contribute to an understanding of the PK characteristics

of pembrolizumab when administered in a Q6W dosing regimen. Safety parameters commonly

used for evaluating investigational systemic anticancer treatments are included as safety

endpoints including, but not limited to, the incidence of, causality, and outcome of adverse

events (AEs)/serious adverse events (SAEs); and changes in vital signs and laboratory values.

AEs will be assessed as defined by National Cancer Institute Common Terminology Criteria for

Adverse Events [NCI CTCAE] Version 4.0).

An objective of this trial is to characterize the PK profile of pembrolizumab

following administration as an IV infusion Q6W. PK data is analyzed after all participants

complete Cycle 5. PK parameters include AUC, Cmax, and Cmin. Formation of Antidrug

Antibodies (ADA) can potentially confound drug exposures at therapeutic doses and prime for

subsequent infusion-related toxicity. Antidrug antibody response to pembrolizumab at the

WO wo 2019/160751 PCT/US2019/017177

beginning of each of Cycles 1, 2, 4, and 5 are determined. Any impact of presence of ADAs on

exposure of pembrolizumab is explored.

This study uses ORR based on RECIST 1.1 criteria as assessed by blinded

independent central review (BICR) as the primary endpoint Objective response rate is an

acceptable measure of clinical benefit for a late stage study that demonstrates superiority of a

new antineoplastic therapy, especially if the magnitude of the effect is large and the therapy has

an acceptable risk/benefit profile. Images are submitted to an imaging CRO (iCRO) and read by

independent central review blinded to treatment assignment to minimize bias in the response

assessments.

Overall survival (OS) is a secondary endpoint and has been recognized as the gold

standard for the demonstration of superiority of a new antineoplastic therapy in randomized

clinical studies. RECIST 1.1 is used by the BICR when assessing images for efficacy measures

and by the local site when determining eligibility. Modified RECIST 1.1 for immune-based

therapeutics (iRECIST) assessment has been developed and published by the RECIST Working

Group, with input from leading experts from industry and academia, along with participation

from the US Food and Drug Administration and the European Medicines Agency. The

unidimensional measurement of target lesions, qualitative assessment of non-target lesions, and

response categories are identical to RECIST 1.1, until progression is seen by RECIST 1.1.

However, if a participant is clinically stable, additional imaging may be performed to confirm

radiographic progression. iRECIST is used by investigators to assess tumor response and

progression and to make treatment decisions as well as for exploratory efficacy analyses where

specified.

Inclusion Criteria

Participants are eligible to be included in the study only if all of the following

criteria apply:

Participant has histologically or cytologically confirmed diagnosis of advanced melanoma

Participant has unresectable Stage III or Stage IV melanoma, as per American Joint

Committee on Cancer (AJCC) staging system not amenable to local therapy.

Participant is untreated for advanced or metastatic disease except as follows: BRAF V600

mutant melanoma may have received standard of care targeted therapy (e.g., BRAF/MEK

inhibitor, alone or in combination) and be eligible for this study

Prior adjuvant or neoadjuvant melanoma therapy is permitted if it was completed at least

4 weeks before randomization and all related AEs have either returned to baseline or

stabilized (resolution of toxic effect(s) of the most recent prior therapy to Grade 1 or less

[except alopecia]). If subject received major surgery or radiation therapy of >30 Gy, they

must have recovered from the toxicity and/or complications from the intervention.

WO wo 2019/160751 PCT/US2019/017177

A female participant is eligible to participate if she is not pregnant, not

breastfeeding, and agrees to follow specific contraceptive guidance during the treatment period

and for at least 120 days or provides informed consent.

A participant should have an Eastern Cooperative Oncology Group (ECOG)

performance status 0 (fully active, able to carry on all pre-disease performance without

restriction) or 1 (restricted in physically strenuous activity but ambulatory and able to carry out

work of a light or sedentary nature, e.g., light house work, office work) and should have adequate

organ function as defined in Table 5. Specimens are collected within 72 hours prior to the start of

study intervention.

Table 5. Adequate Organ Function Laboratory Values

System Laboratory Value

Hematological

Absolute neutrophil count (ANC) > : 1500/uL

Platelets 100 000/uL

Hemoglobin > 9.0 g/dL or > 5.6 mmol/L¹

Renal

Creatinine OR < 1.5 X ULN OR Measured or calculated2 creatinine > 30 mL/min for participant with creatinine clearance levels >1.5 X institutional ULN (GFR can also be used in place of creatinine or CrCl)

Hepatic

Total bilirubin < 1.5 X ULN OR direct bilirubin < ULN for participants with total bilirubin levels > 1.5 X

ULN AST (SGOT) and ALT (SGPT) <2.5 X ULN (<5 X ULN for participants with liver metastases)

Coagulation

International normalized ratio (INR) OR <1.5 X ULN unless participant is receiving prothrombin time (PT) anticoagulant therapy as long as PT or PTT is

Activated partial thromboplastin time within therapeutic range of intended use of anticoagulants (aPTT) 1 Criteria must be met without erythropoietin dependency and without packed red blood cell (pRBC) transfusion within last 2 weeks.

2 Creatinine clearance (CrCl) should be calculated per institutional standard.

ALT (SGPT)=alanine aminotransferase (serum glutamic pyruvic transaminase); AST (SGOT)=aspartate aminotransferase (serum glutamic oxaloacetic transaminase); GFR=glomerular filtration rate; ULN=upper limit of normal.

Exclusion Criteria

Participants are excluded from the study if any of the following criteria apply:

The participant is a woman of child-bearing potential (WOCBP) who has a positive urine

pregnancy test within 72 hours prior to randomization or treatment allocation. If the urine

test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

The participant has received prior systemic treatment for unresectable or metastatic

melanoma (except as noted in inclusion criteria described above).

The participant has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2

or with an agent directed to another stimulatory or co-inhibitory T-cell receptor (e.g., OX-

40 and CD137) or any other antibody or drug specifically targeting checkpoint pathways

other than anti-CTLA-4 which is permitted in the adjuvant setting.

The participant has received prior radiotherapy within 2 weeks of start of study treatment.

Participants must have recovered from all radiation-related toxicities, not require

corticosteroids, and not have had radiation pneumonitis.

The participant has received a live vaccine within 30 days prior to the first dose of study

drug. Examples of live vaccines include, but are not limited to, the following: measles,

mumps, rubella, varicella/zosten (chicken pox), yellow fever, rabies, Bacillus

Calmette-Guérin (BCG), and typhoid vaccine. Seasonal influenza vaccines for injection

are generally killed virus vaccines and are allowed; however, intranasal influenza

vaccines (e.g., FluMist®) are live attenuated vaccines and are not allowed.

The participant is currently participating in or has participated in a study of an

investigational agent or has used an investigational device within 4 weeks prior to the

first dose of study intervention.

The participant has a diagnosis of immunodeficiency or is receiving chronic systemic

steroid therapy (in dosing exceeding 10 mg daily of prednisone equivalent) or any other

form of immunosuppressive therapy within 7 days prior the first dose of study drug.

The participant has a known additional malignancy that is progressing or has required

active treatment within the past 2 years. Note: Participants with basal cell carcinoma of

the skin, squamous cell carcinoma of the skin, or carcinoma in situ (e.g., breast

carcinoma, cervical cancer in situ) that have undergone potentially curative therapy are

not excluded.

The participant has known active CNS metastases and/or carcinomatous meningitis.

Participants with previously treated brain metastases may participate provided they are

radiologically stable, (i.e., without evidence of progression) for at least 4 weeks by repeat

imaging (note that the repeat imaging should be performed during study screening),

clinically stable and without requirement of steroid treatment for at least 14 days prior to

first dose of study intervention.

The participant has severe hypersensitivity (> Grade 3) to pembrolizumab and/or any of

its excipients.

The participant has ocular melanoma.

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The participant has an active autoimmune disease that has required systemic treatment in

past 2 years (i.e., with use of disease modifying agents, corticosteroids or

immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or physiologic

corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered

a form of systemic treatment and is allowed.

The participant has a history of (non-infectious) pneumonitis that required steroids or has

current pneumonitis.

The participant has an active infection requiring systemic therapy.

The participant has a known history of human immunodeficiency virus (HIV) infection.

The participant has a known history of Hepatitis B (defined as Hepatitis B surface antigen

[HBsAg] reactive) or known active Hepatitis C virus (defined as HCV RNA [qualitative]

is detected) infection.

The participant has a history or current evidence of any condition, therapy, or laboratory

abnormality that might confound the results of the study, interfere with the participant's

participation for the full duration of the study, or is not in the best interest of the

participant to participate, in the opinion of the treating investigator.

The participant has a known psychiatric or substance abuse disorder that would interfere

with cooperating with the requirements of the study.

The participant is pregnant or breastfeeding or expecting to conceive or father children

within the projected duration of the study, starting with the screening visit through 120

days after the last dose of study intervention.

Discontinuation of Study Intervention and Participant Withdrawal

Discontinuation of study intervention does not represent withdrawal from the

study. As certain data on clinical events beyond study intervention discontinuation may be

important to the study, they must be collected through the participant's last scheduled follow-up,

even if the participant has discontinued study intervention. Therefore, all participants who

discontinue study intervention prior to completion of the protocol-specified treatment period will

still continue to participate in the study.

Participants may discontinue study intervention at any time for any reason or be

dropped from the study intervention at the discretion of the investigator should any untoward

effect occur. In addition, a participant may be discontinued from study intervention by the

investigator if study intervention is inappropriate, the study plan is violated, or for administrative

and/or other safety reasons.

A participant must be discontinued from study intervention but continue to be

monitored in the study for any of the following reasons:

The participant or participant's legally acceptable representative requests to discontinue

study intervention.

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The participant interrupts study intervention administration for more than 12 consecutive

weeks or has 3 cumulative missed doses.

The participant has a medical condition or personal circumstance which, in the opinion of

the investigator, placed the participant at unnecessary risk from continued administration

of study intervention.

The participant has a confirmed positive serum pregnancy test.

The participant has confirmed radiographic disease progression

The participant has any progression or recurrence of any malignancy, or any occurrence

of another malignancy that requires active treatment

The participant has unacceptable adverse experiences.

The participant has intercurrent illness other than another malignancy as noted above that

prevents further administration of treatment.

Investigator decides to discontinue treatment.

The participant has recurrent Grade 2 pneumonitis

The participant has completed 35 treatments (approximately 2 years) with

pembrolizumab A participant is withdrawn from the study if the participant or participant's legally

acceptable representative withdraws consent from the study. If a participant withdraws from the

study, they will no longer receive study treatment or be followed at scheduled protocol visits.

Informed Consent The investigator or medically qualified designee obtains documented consent

from each potential participant or each participant's legally acceptable representative prior to

participating in a clinical study. If there are changes to the participant's status during the study

(e.g., health or age of majority requirements), the investigator or medically qualified designee

ensures the appropriate consent is in place.

Efficacy/Assessments

Tumor assessments include all known or suspected disease sites. Imaging may

include chest, abdomen, and pelvis computed tomography (CT) or magnetic resonance imaging

(MRI) at baseline and when disease progression or brain metastases is suspected. Tumor imaging

is strongly preferred to be acquired by CT. For chest, abdomen and pelvis, contrast-enhanced

MRI may be used when CT with iodinated contrast is contraindicated, or when mandated by

local practice. For the brain, MRI is the strongly preferred imaging modality.

The same imaging modality technique (ideally the same scanner, and consistent

use of contrast) is used in a participant throughout the study. Consistent use of imaging

techniques will help to optimize the reproducibility of the assessment of existing and new tumor

burden, and to improve the accuracy of the assessment of response or progression. All scheduled

images for all study participants are reviewed by the investigator for disease progression. In

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addition, images (including those obtained via other modalities) that are obtained at an

unscheduled time point to determine disease progression (as well as imaging obtained for other

reasons, but that capture radiologic progression based on investigator assessment), are also be

filed at the study site.

Confirmation of measurable disease based on RECIST 1.1 by BICR at screening

will be used to determine participant eligibility. Confirmation by the BICR that the participant's

imaging shows at least 1 lesion that is appropriate for selection as a target lesion per RECIST 1.1

is required prior to participant allocation.

Initial Tumor Imaging

Initial tumor imaging at screening is performed within 28 days prior to the date of

first dose. Any imaging obtained after Cycle 1 Day 1 of treatment is not included in the

screening assessment. The site study team reviews screening images to confirm the participant

has measurable disease per RECIST 1.1. If brain imaging is performed to document the stability

of existing metastases, MRI is used if possible. If MRI is medically contraindicated, CT with

contrast is an acceptable alternative.

Tumor Imaging During the Study

The first on-study imaging assessment is performed at 12 weeks (84 days

7 days]) from the date of first dose. Subsequent tumor imaging is performed every 9 weeks

(63 days +7 days) or more frequently if clinically indicated. After 52 weeks (365 days 7 days),

participants who remain on treatment will have imaging performed every 12 weeks (84 days +7

days).

Objective response is confirmed by a repeat imaging assessment. Tumor imaging

to confirm PR or CR is performed at least 4 weeks after the first indication of a response is

observed. Participants will then return to regular scheduled imaging, starting with the next

scheduled imaging time point. Participants who receive additional imaging for confirmation do

not need to undergo the next scheduled tumor imaging if it is less than 4 weeks later; tumor

imaging may resume at the subsequent scheduled imaging time point.

Per modified iRECIST, disease progression is confirmed by the site 4 to 8 weeks

after first radiologic evidence of progressive disease (PD) in clinically stable participants.

Participants who have unconfirmed disease progression may continue on treatment at the

discretion of the investigator until progression is confirmed by the site. Participants who receive

confirmatory imaging do not need to undergo the next scheduled tumor imaging if it is less than

4 weeks later; tumor imaging may resume at the subsequent scheduled imaging time point, if

clinically stable. Participants who have confirmed disease progression by iRECIST, as assessed

by the site, will discontinue study treatment.

End-of-Treatment and Follow-up Tumor Imaging

42

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For participants who discontinue study intervention, tumor imaging is performed

at the time of treatment discontinuation (+4 week window). If previous imaging was obtained

within 4 weeks prior to the date of discontinuation, then imaging at treatment discontinuation is

not mandatory. For participants who discontinue study intervention due to documented disease

progression, this is the final required tumor imaging if the investigator elects not to implement

iRECIST. For participants who discontinue study intervention without documented disease

progression, every effort should be made to continue monitoring disease status by tumor imaging

using the same imaging schedule used while on treatment every 12 weeks (+7 days) until the start

of a new anticancer treatment, disease progression, pregnancy, death, withdrawal of consent, or

the end of the study, whichever occurs first.

RECIST 1.1 Assessment of Disease

RECIST 1.1 is used as the primary measure for assessment of tumor response,

date of disease progression, and as a basis for all protocol guidelines related to disease status

(e.g., discontinuation of study intervention). Although RECIST 1.1 references a maximum of 5

target lesions in total and 2 per organ, this protocol allows a maximum of 10 target lesions in

total and 5 per organ, if clinically relevant to enable a broader sampling of tumor burden.

iRECIST Assessment of Disease

iRECIST is based on RECIST 1.1, but adapted to account for the unique tumor

response seen with immunotherapeutic drugs. iRECIST will be used by the investigator to assess

tumor response and progression, and make treatment decisions. When clinically stable,

participants are not discontinued until progression is confirmed by the investigator, working with

local radiology. This allowance to continue treatment despite initial radiologic PD takes into

account the observation that some participants can have a transient tumor flare in the first few

months after the start of immunotherapy, and then experience subsequent disease response.

Any participant deemed clinically unstable is discontinued from study

intervention at the time when site-assessed first radiologic evidence of PD, and is not required to

have repeat tumor imaging for confirmation of PD by iRECIST. If the investigator decides to

continue treatment, the participant may continue to receive study intervention and the tumor

assessment should be repeated 4 to 8 weeks later to confirm PD by iRECIST, per investigator

assessment. If repeat imaging does not confirm PD per iRECIST, as assessed by the investigator,

and the participant continues to be clinically stable, study intervention continues and follows the

regular imaging schedule. If PD is confirmed, participants are discontinued from study

intervention.

If a participant has confirmed radiographic progression (iCPD), study intervention

is discontinued; however, if the participant is achieving a clinically meaningful benefit, an

exception to continue study intervention is considered. In this case, if study intervention is

43 continued, tumor imaging continues to be performed. A summary of imaging and treatment requirements after first radiologic evidence of progression is provided in Table 6.

Table 6 Imaging and Treatment after First Radiologic Evidence of Progressive Disease Clinically Stable Clinically Unstable Imaging Treatment Imaging Treatment First radiologic Repeat May continue Repeat imaging Discontinue evidence of PD by imaging at 4 study treatment at 4 to 8 weeks treatment RECIST 1.1 per to 8 weeks to at the to confirm PD investigator confirm PD assessment of per assessment the investigator investigator's and after the discretion only.

participant's

consent First radiologic Repeat May continue Repeat imaging Discontinue evidence of PD by imaging at 4 study at 4 to 8 weeks treatment RECIST 1.1 to 8 weeks to intervention at to confirm PD confirm PD. the per investigator's investigator's

discretion while discretion only.

awaiting confirmatory tumor imaging by site by

iRECIST. Repeat tumor No additional Discontinue No additional Not applicable imaging confirms imaging treatment. imaging PD (iCPD) by required. required.

iRECIST per investigator

assessment. Repeat tumor Repeat Continue study Repeat imaging Discontinue imaging shows imaging at 4 intervention at at 4 to 8 weeks treatment iUPD by iRECIST to 8 weeks to the to confirm PD per investigator confirm PD. investigator's per assessment. May occur at discretion. investigator's next regularly discretion only.

scheduled imaging visit.

Repeat tumor Continue Continue study Continue May restart imaging shows regularly intervention at regularly study iSD, iPR, or iCR by scheduled the scheduled intervention if

iRECIST per imaging investigator's imaging condition has investigator assessments. discretion. assessments. improved assessment. and/or clinically stable

per investigator's

discretion. Next tumor imaging

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Clinically Stable Clinically Unstable Imaging Treatment Imaging Treatment should occur according to the regular imaging schedule.

Abbreviations: iCPD=iRECIST confirmed progressive disease; iCR=iRECIST complete response; iPR=iRECIST confirmed partial response; iRECIST=modified Response Evaluation Criteria in Solid Tumors 1.1 for immune-based therapeutics; iSD=iRECIST stable disease; iUPD=iRECIST unconfirmed progressive disease; PD=progressive disease; RECIST 1.1=Response Evaluation Criteria in Solid Tumors 1.1; VOP=verification of progression

Safety Assessments

Safety assessments include the collection of AEs and SAEs, monitoring of vital

signs and laboratory assessments (including pregnancy tests), performance of electrocardiograms

(ECGs) and physical examinations, and verification of concurrent medications.

Adverse Events

The investigator or qualified designee assesses each subject to evaluate for

potential new or worsening AEs and more frequently if clinically indicated. Assessment of AEs

includes, but is not limited to, the type, incidence, severity (graded by the National Cancer

Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] Version 4.0), timing,

seriousness, and relatedness to study drug. Adverse events that occur during the study, including

baseline signs and symptoms, are recorded.

Full Physical Examination

The investigator or qualified designee performs a complete physical exam during

the Screening period. Clinically significant abnormal findings are recorded as medical history.

After the first dose of study intervention, new clinically significant abnormal findings are

recorded as AEs.

Directed Physical Examination

For cycles that do not require a full physical exam, the investigator or qualified

designee performs a directed physical exam as clinically indicated prior to the administration of

the study intervention. New clinically significant abnormal findings are recorded as AEs.

Vital Signs

Vital signs are measured in a semi-supine position after 5 minutes rest and include

temperature, systolic and diastolic blood pressure, respiratory rate, pulse rate, and weight. Height

is collected at screening only.

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Electrocardiograms

A standard 12-lead ECG is performed using local standard procedures. Clinically

significant abnormal findings at Screening are recorded as medical history. Additional ECG(s)

are performed on study when clinically necessary. Clinically significant findings seen on the

follow-up ECGs are recorded as AEs.

Clinical Safety Laboratory Assessments

The tests detailed in Table 7are performed by a local laboratory. Additional tests

may be performed at any time during the study as determined necessary by the investigator.

Table 7 Protocol-Required Safety Laboratory Assessments Laboratory Parameters Assessments Hematology Platelet Count RBC Indices: WBC count with Differential: RBC Count MCV Hemoglobin Neutrophils MCH %Reticulocytes Lymphocytes Hematocrit Monocytes Eosinophils Basophils Chemistry Blood Urea Potassium Aspartate Total bilirubin

Nitrogen (BUN) Aminotransferase (and direct bilirubin, if total (AST)/ Serum Glutamic- bilirubin is

Oxaloacetic elevated above Transaminase the upper limit of

(SGOT) normal)

Albumin Bicarbonate Chloride Phosphorous Creatinine Sodium Alanine Total Protein Aminotransferase (ALT)/ Serum Glutamic- Pyruvic Transaminase (SGPT) Glucose Calcium Alkaline TSH phosphatase Total T3 (or free

T3) Total T4 (or free

T4)a Routine Specific gravity

Urinalysis pH, glucose, protein, blood, ketones, [bilirubin, urobilinogen, nitrite, leukocyte esterase] by dipstick

Microscopic examination (if blood or protein is abnormal) Other Follicle-stimulating hormone and estradiol (as needed in women of non- Screening childbearing potential only)

Tests [Serum or urine] [alcohol and drug screen (to include at minimum:

WO wo 2019/160751 PCT/US2019/017177

amphetamines, barbiturates, cocaine, opiates, cannabinoids and benzodiazepines) if applicable]

[Serum or urine] B-human chorionic gonadotropin (B-hCG) pregnancy test (as needed for WOCBP)

[Serology [(HIV antibody, hepatitis B surface antigen [HBsAg], and hepatitis C virus antibody)] or specify other tests] [if applicable]

NOTES: aT3 and T4 are preferred; if not available, free T3 and free T4 may be tested.

Abbreviations: B-hCG=3-human chorionic gonadotropin; ALT=alanine transaminase; AST=aspartate transaminase; BUN=blood urea nitrogen; HBsAg=hepatitis B surface antigen; HIV=human immunodeficiency virus; MCH=mean corpuscular hemoglobin; MCV=mean corpuscular volume; RBC=red blood cell; SGOT=serum glutamic oxaloacetic transaminase; SGPT=serum glutamic pyruvic transaminase; TSH=thyroid stimulating hormone; WBC=white blood cell; WOCBP=woman/women of childbearing potential.

Time Period and Frequency for Collecting AE, SAE, and Other Reportable Safety Event

Information

All AEs, SAEs, and other reportable safety events that occur after the consent

form is signed but before treatment allocation/randomization must be reported by the investigator

if the participant is receiving placebo run-in or other run-in treatment, if the event cause the

participant to be excluded from the study, or is the result of a protocol-specified intervention,

including but not limited to washout or discontinuation of usual therapy, diet, or a procedure. All

AEs from the time of treatment allocation/randomization through 30 days following cessation of

study intervention must be reported by the investigator.

All AEs meeting serious criteria, from the time of treatment

allocation/randomization through 90 days following cessation of study intervention or 30 days

following cessation of study intervention if the participant initiates new anticancer therapy,

whichever is earlier, must be reported by the investigator. Additionally, any SAE brought to the

attention of an investigator at any time outside of the time period specified above is reported

immediately if the event is considered drug-related.

Statistical Methods for Efficacy Analyses

Objective Response Rate (ORR) - ORR is calculated as the ratio of the number of

participants reported to have achieved a confirmed CR or PR verified by BICR, divided by the

number of participants included in APaT population. Participants in the APaT analysis

population without ORR assessments will be counted as non-responders. A 95% exact binomial

CI (based on method Clopper and Pearson, 1934) is calculated for the true ORR.

Progression-Free Survival (PFS)- The non-parametric Kaplan-Meier method is

used to estimate the PFS distribution. 95% CIs for the median PFS and PFS point estimates at

various follow-up times from first day of study treatment will be calculated. Since disease

progression is assessed periodically, PD can occur any time in the time interval between the last

assessment where PD was not documented and the assessment when PD is documented. The true

WO wo 2019/160751 PCT/US2019/017177

date of PD will be approximated by the date of the first assessment at which PD is objectively

documented based on RECIST 1.1 by BICR. Death is always considered as a PFS event.

Participants who do not experience a PFS event will be censored at the last disease assessment.

For the analysis of PFS, if the events (PD or death) are immediately after more than one missed

disease assessment, the data are censored at the last disease assessment prior to missing visits.

Also, data after new anticancer therapy are censored at the last disease assessment prior to the

initiation of new anticancer therapy. If a participant meets multiple criteria for censoring, the

censoring criterion that occurs earliest will be applied.

Overall Survival (OS)- The non-parametric Kaplan-Meier method is used to

estimate the os distribution. 95% CIs for the median os and os point estimates at various

follow-up times from first day of study treatment is calculated.

Duration of Response (DOR) - DOR is summarized descriptively using the non-

parametric Kaplan-Meier method. Only the subset of participants who show a CR or PR are

included in this analysis.

Analysis Strategy for Key Efficacy Endpoint

Table 8 summarizes the primary analysis approach for key efficacy endpoints.

Table 8. Analysis Strategy for Key Efficacy Endpoints Analysis Missing Data Endpoint Statistical Method Population Approach Primary Endpoints Participants without Exact method based assessments are on binomial considered ORR per RECIST 1.1 distribution APaT non-responders and by BICR (Clopper-Pearson conservatively method) included in the

denominator Key Secondary Endpoint Summary statistics Primary censoring PFS per RECIST 1.1 using Kaplan-Meier rule APaT by BICR method Summary statistics Censored at the last using Kaplan-Meier APaT os known alive date method Non-responders are Summary statistics excluded from DOR per RECIST 1.1 using Kaplan-Meier analysis.

method APaT Responders are by BICR censored according to the censoring rules.

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Analysis Missing Data Endpoint Statistical Method Population Approach a Statistical models are described in further detail in the text.

Abbreviations: APaT=All Participants as Treated; BICR=blinded independent central review; DOR=duration of response; ORR=objective response rate; OS=overall survival; PFS-progression-free survival; RECIST=Response Evaluation Criteria in Solid Tumors

Statistical Methods for Safety Analyses

Safety and tolerability are assessed by clinical review of all relevant parameters

including adverse experiences and laboratory parameters. The broad AE categories consisting of

the percentage of participants with any AE, a drug-related AE, a serious AE, an AE which is both

drug-related and serious, and who discontinued due to an AE are summarized via point estimates

with 95% CIs (Table 9).

Table 9. Analysis Strategy for Safety Parameters

Within Group Safety Endpoint 95% CI Descriptive Statistics

Any AE X X Any Serious AE X X Any Drug-related AE X X Any Serious and Drug-related AE X Discontinuation due to AE X Specific AEs, SOCs, or PDLCs Change from Baseline Results (Labs, Vital Signs) X X Note: 95% CIs will be calculated using the Clopper Pearson method X = results are provided Abbreviations: SOC=System Organ Class; PDLC=Pre-Defined Limit of Change

An AE is any untoward medical occurrence in a clinical study participant,

temporally associated with the use of study intervention, whether or not considered related to the

study intervention. An AE can therefore be any unfavorable and unintended sign (including an

abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated

with the use of the drug. The following are included as AEs:

Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or

other safety assessments (e.g., ECG, radiological scans, vital signs measurements),

including those that worsen from baseline, or are considered clinically significant in the

medical and scientific judgment of the investigator.

Exacerbation of a chronic or intermittent pre-existing condition including either an

increase in frequency and/or intensity of the condition.

New conditions detected or diagnosed after study intervention administration even though

it may have been present before the start of the study.

Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction.

Signs, symptoms, or the clinical sequelae of a suspected overdose of either study

intervention or a concomitant medication.

Worsening of signs and symptoms of malignancy during the study is reported as an AE.

Disease progression assessed by measurement of malignant lesions on radiographs or

other methods are not be reported as an AE, unless the event results in hospitalization or

death.

The following events do not meet the AE definition for purposes of this study:

Medical or surgical procedure (e.g., endoscopy, appendectomy): the condition that leads

to the procedure is the AE.

Situations in which an untoward medical occurrence did not occur (social and/or

convenience admission to a hospital).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or

detected at the start of the study that do not worsen.

Surgery planned prior to informed consent to treat a pre-existing condition that has not

worsened.

If an event is not an AE per definition above, then it cannot be an SAE even if

serious conditions are met. An SAE is defined as any untoward medical occurrence that, at any

dose:

Results in death

Is life-threatening. The term "life-threatening" in the definition of "serious" refers to an

event in which the participant was at risk of death at the time of the event. It does not

refer to an event, which hypothetically might have caused death, if it were more severe.

Requires inpatient hospitalization or prolongation of existing hospitalization.

Hospitalization is defined as an inpatient admission, regardless of length of stay, even if

the hospitalization is a precautionary measure for continued observation. Hospitalization

for an elective procedure to treat a pre-existing condition that has not worsened is not an

SAE. A pre-existing condition is a clinical condition that is diagnosed prior to the use of

an MSD product and is documented in the participant's medical history.

Results in persistent or significant disability/incapacity. The term disability means a

substantial disruption of a person's ability to conduct normal life functions. This

definition is not intended to include experiences of relatively minor medical significance

such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental

trauma (e.g., sprained ankle) that may interfere with or prevent everyday life functions but

do not constitute a substantial disruption.

Is a congenital anomaly/birth defect in offspring of participant taking the product

regardless of time to diagnosis.

Medical or scientific judgment is exercised in deciding whether SAE reporting is

appropriate in other situations such as important medical events that may not be immediately - 50

WO wo 2019/160751 PCT/US2019/017177

life-threatening or result in death or hospitalization but may jeopardize the participant or may

require medical or surgical intervention to prevent one of the other outcomes listed in the above

definition. These events are usually considered serious. Examples of such events include

invasive or malignant cancers, intensive treatment in an emergency room or at home for allergic

bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization, or

development of drug dependency or drug abuse.

Demographics and Baseline Characteristics

The number and percentage of subjects screened, allocated, the primary reasons

for screening failure, and the primary reasons for discontinuation are displayed. Demographic

variables (e.g., age, gender), baseline characteristics, primary and secondary diagnoses, and prior

and concomitant therapies is summarized either by descriptive statistics or categorical tables for

all enrolled subjects.

Subgroup Analyses To determine whether the response rate is consistent across various subgroups, the

estimate of the response rate (with a nominal 95% CI) for the primary endpoint is estimated

within each category of the following classification variables:

Age category (<65 VS. >65 years)

Sex (female VS. male)

Race (white VS. non-white)

Disease stage (III VS. IVM1 VS. IVM1b VS IVM1c)

Brain metastasis (yes VS. no)

ECOG status (0 VS. 1)

PD-L1 status (positive VS. negative)

BRAF wild type versus BRAF mutant (no prior treatment) versus BRAF mutant (prior

treatment)

A Forest plot is produced, which provides the estimated point estimates and CIs for the treatment

effect across the categories of subgroups listed above. Any specified subgroups that have less

than 10 participants are excluded from analysis.

All references cited herein are incorporated by reference to the same extent as if

each individual publication, database entry (e.g. Genbank or GeneID entries), patent application,

or patent, was specifically indicated to be incorporated by reference. This statement is intended

by Applicants, pursuant to 37 C.F.R. $1.57(b)(1), to relate to each and every individual

publication, database entry (e.g. Genbank or GeneID entries), patent application, or patent, each

of which is clearly identified in compliance with 37 C.F.R. $1.57(b)(2), even if such citation is

not immediately adjacent to a dedicated statement of incorporation by reference. Citation of the

references herein is not intended as an admission that the reference is pertinent prior art, nor does

it constitute any admission as to the contents or date of these publications or documents.

Claims (22)

Claims:

1. A method of treating cancer in a human patient comprising administering about 400 mg of pembrolizumab to the patient every approximately six weeks.

2. Use of about 400 mg of pembrolizumab in the manufacture of a medicament for 2019220495

treating cancer in a human patient, wherein the medicament is administered to the patient every approximately six weeks.

3. The method of claim 1 or use of claim 2, wherein the cancer is selected from the group consisting of: melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, endometrial cancer, squamous cell carcinoma, or salivary cancer.

4. The method of claim 1 or the use of claim 2, wherein the patient has a tumor with a high mutational burden.

5. The method of claim 1 or the use of claim 2, wherein the patient has a microsatellite instability-high (MSI-H) or mismatch repair deficient solid tumor.

6. The method of claim 1 or the use of claim 2, wherein the cancer is unresectable or metastatic melanoma.

7. The method of claim 1 or the use of claim 2, wherein the cancer is metastatic non- small cell lung cancer (NSCLC).

8. The method of claim 1 or the use of claim 2, wherein the cancer is recurrent or metastatic head and neck squamous cell cancer (HNSCC).

9. The method of claim 1 or the use of claim 2, wherein the cancer is (1) refractory classical Hodgkin lymphoma (cHL), or (2) cHL and the patient has relapsed after 3 or more lines of therapy for cHL.

10. The method of claim 1 or the use of claim 2, wherein the cancer is locally advanced

or metastatic urothelial carcinoma.

11. The method of claim 1 or the use of claim 2, wherein the cancer is locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

12. The method of claim 1 or the use of claim 2, wherein the cancer is cervical cancer. 2019220495

13. The method of claim 1 or the use of claim 2, wherein the cancer is primary mediastinal large B-cell lymphoma (PMBCL).

14. The method of claim 1 or the use of claim 2, wherein the cancer is resected high-risk stage III melanoma.

15. The method of claim 1 or the use of claim 2, wherein the cancer is hepatocellular carcinoma.

16. The method of claim 1 or the use of claim 2, wherein the cancer is renal cell carcinoma (RCC).

17. The method of claim 1 or the use of claim 2, wherein the cancer is recurrent, locally advanced or metastatic Merkel cell carcinoma (MCC).

18. A composition comprising about 400 mg of pembrolizumab and a pharmaceutically acceptable carrier when used approximately every six weeks for the treatment of cancer.

19. The composition of claim 18, further comprising 10 mM histidine, pH 5.5, 7% sucrose, and 0.02% polysorbate 80.

20. Use of the composition of any one of claims 18-19 for treating an individual suffering from cancer.

21. The use of claim 20, wherein the cancer is melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, endometrial cancer, squamous cell carcinoma, or salivary cancer.

22. The method of claim 1 or the use of claim 2, wherein the cancer is endometrial cancer.